Tolerogenic synthetic nanocarriers coupled to cd1d-restricted antigens and methods of use

ABSTRACT

Disclosed are synthetic nanocarrier compositions, and related methods, comprising CD1d-restricted antigens and immunosuppressants that provide tolerogenic immune responses.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S.provisional application 61/480,946, filed Apr. 29, 2011, 61/513,514,filed Jul. 29, 2011, 61/531,147, filed Sep. 6, 2011, 61/531,153, filedSep. 6, 2011, 61/531,164, filed Sep. 6, 2011, 61/531,168, filed Sep. 6,2011, 61/531,175, filed Sep. 6, 2011, 61/531,180, filed Sep. 6, 2011,61/531,194, filed Sep. 6, 2011, 61/531,204, filed Sep. 6, 2011,61/531,209, filed Sep. 6, 2011, 61/531,215, filed Sep. 6, 2011, theentire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to synthetic nanocarrier compositions withCD1d-restricted antigens and immunosuppressants, and related methods.The compositions and methods allow for the stimulation of tolerogenicimmune responses in a subject via iNKT cells. The tolerogenic immuneresponses can be antigen-specific, and the compositions provided mayalso include APC presentable antigens. Such compositions allow forefficient uptake by APCs to shift the immune response in favor oftolerogenic immune response development.

BACKGROUND OF THE INVENTION

Conventional immunosuppressant drugs are broad-acting. Additionally, inorder to maintain immunosuppression, immunosuppressant drug therapy isgenerally a life-long proposition. Unfortunately, the use ofbroad-acting immunosuppressants are associated with a risk of severeside effects, such as tumors, infections, nephrotoxicity and metabolicdisorders. Accordingly, new immunosuppressant therapies would bebeneficial.

SUMMARY OF THE INVENTION

In one aspect, a composition comprising (i) a first population ofsynthetic nanocarriers coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers coupled to CD1d-restricted antigensis provided. In one embodiment, the first population and secondpopulation are the same population. In another embodiment, the firstpopulation and second population are different populations.

In another embodiment, the immunosuppressants comprise a statin, an mTORinhibitor, a TGF-β signaling agent, a corticosteroid, an inhibitor ofmitochondrial function, a P38 inhibitor, an NF-κβ inhibitor, anadenosine receptor agonist, a prostaglandin E2 agonist, aphosphodiesterasse 4 inhibitor, an HDAC inhibitor or a proteasomeinhibitor. In another embodiment, the mTOR inhibitor is rapamycin or arapamycin analog.

In another embodiment, the CD1d-restricted antigens compriseglycolipids. In another embodiment, the CD1d-restricted antigenscomprise α-galactosylceramide, β-glucosylceramide, α-linkedglycosphingolipid from Sphingomonas spp., galactosyl diaglycerol fromBorrelia burgdorferi, lypophosphoglycan from Leishmania orphosphatidylinositol tetramannoside from Mycobacterium leprae.

In another embodiment, the composition is in an amount effective togenerate a tolerogenic immune response when administered to a subject.In one embodiment, the tolerogenic immune response is the stimulation ofiNKT cells. In another embodiment, the tolerogenic immune response isthe production of IL-4 by iNKT cells. In another embodiment, thetolerogenic immune response is the production of IL-10 by iNKT cells. Inanother embodiment, the tolerogenic immune response is iNKT cellstimulation. In another embodiment, the tolerogenic immune response isiNKT cell anergy or the reduction in pathogenic iNKT cells. In anotherembodiment, the tolerogenic immune response is a reduction in IFN-γproduced by iNKT cells. In another embodiment, the tolerogenic immuneresponse is antigen-specific.

In another embodiment, the composition further comprises APC presentableantigens. In another embodiment, the APC presentable antigens arecoupled to the first and/or second populations of syntheticnanocarriers. In another embodiment, the APC presentable antigens arecoupled to a third population of synthetic nanocarriers. In oneembodiment, the coupling is covalent coupling. In another embodiment,the coupling is noncovalent coupling. In another embodiment, the APCpresentable antigens are not coupled to synthetic nanocarriers.

In another embodiment, the APC presentable antigens comprise MHC ClassI-restricted, MHC Class II-restricted epitopes and/or B cell epitopes.In yet another embodiment, the APC presentable antigens comprisesubstantially no B cell epitopes. In one embodiment, the APC presentableantigens are or are obtained or derived from proteins, polypeptides,polysaccharides, polynucleotides or cells.

In a further embodiment, the composition further comprises atransplantable graft.

In another embodiment, the APC presentable antigens are autoantigens,allergens, or are associated with an inflammatory disease, fatty liverdisease, an autoimmune disease, allergy, sickle cell disease,spontaneous abortion organ or tissue rejection or graft versus hostdisease.

In another embodiment, the load of the immunosuppressants and/orCD1d-restricted antigens on average across the first and/or secondpopulation of synthetic nanocarriers is between 0.0001% and 50%. Inanother embodiment, the load of the immunosuppressants and/orCD1d-restricted antigens on average across the first and/or secondpopulation of synthetic nanocarriers is between 0.1% and 10%.

In another embodiment, the synthetic nanocarriers of the firstpopulation and/or second population and/or third population compriselipid nanoparticles, polymeric nanoparticles, metallic nanoparticles,surfactant-based emulsions, dendrimers, buckyballs, nanowires,virus-like particles or peptide or protein particles. In anotherembodiment, the synthetic nanocarriers of the first and/or second and/orthird populations comprise lipid nanoparticles. In another embodiment,the synthetic nanocarriers of the first and/or second and/or thirdpopulations comprise liposomes. In another embodiment, the syntheticnanocarriers of the first and/or second and/or third populationscomprise metallic nanoparticles. In another embodiment, the metallicnanoparticles comprise gold nanoparticles. In another embodiment, thesynthetic nanocarriers of the first and/or second and/or thirdpopulations comprise polymeric nanoparticles. In another embodiment, thepolymeric nanoparticles comprise polymer that is anon-methoxy-terminated, pluronic polymer. In another embodiment, thepolymeric nanoparticles comprise a polyester, a polyester coupled to apolyether, polyamino acid, polycarbonate, polyacetal, polyketal,polysaccharide, polyethyloxazoline or polyethyleneimine. In anotherembodiment, the polyester comprises a poly(lactic acid), poly(glycolicacid), poly(lactic-co-glycolic acid) or polycaprolactone. In anotherembodiment, the polymeric nanoparticles comprise a polyester and apolyester coupled to a polyether. In another embodiment, the polyethercomprises polyethylene glycol or polypropylene glycol.

In another embodiment, the mean of a particle size distribution obtainedusing dynamic light scattering of the synthetic nanocarriers of thefirst and/or second and/or third population is a diameter greater than100 nm. In another embodiment, the diameter is greater than 150 nm. Inanother embodiment, the diameter is greater than 200 nm. In anotherembodiment, the diameter is greater than 250 nm. In another embodiment,the diameter is greater than 300 nm.

In another embodiment, the aspect ratio of the synthetic nanocarriers ofthe first population and/or second population and/or third population isgreater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.

In another embodiment, the composition further comprises apharmaceutically acceptable excipient.

In another aspect, a dosage form comprising any of the compositionsprovided herein is provided.

In another aspect, a method comprising administering any of thecompositions or dosage forms to a subject is provided.

In another aspect, a method comprising administering to a subject acomposition comprising (i) a first population of synthetic nanocarrierscoupled to immunosuppressants, and (ii) a second population of syntheticnanocarriers coupled to CD1d-restricted antigens, wherein thecomposition is in an amount effective to generate a tolerogenic immuneresponse in the subject is provided. In another aspect, a methodcomprising generating a tolerogenic immune response in a subject byadministering a composition comprising (i) a first population ofsynthetic nanocarriers coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers coupled to CD1d-restricted antigensis provided. In another aspect, a method comprising administering acomposition to a subject according to a protocol that was previouslyshown to generate a tolerogenic immune response in one or more testsubjects; wherein the composition comprises (i) a first population ofsynthetic nanocarriers coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers coupled to CD1d-restricted antigensis provided

In one embodiment, the first population and second population are thesame population. In another embodiment, the first population and secondpopulation are different populations.

In another embodiment, the method further comprises providing oridentifying the subject.

In another embodiment, the immunosuppressants comprise a statin, an mTORinhibitor, a TGF-β signaling agent, a corticosteroid, an inhibitor ofmitochondrial function, a P38 inhibitor, an NF-κβ inhibitor, anadenosine receptor agonist, a prostaglandin E2 agonist, aphosphodiesterasse 4 inhibitor, an HDAC inhibitor or a proteasomeinhibitor. In another embodiment, the mTOR inhibitor is rapamycin or arapamycin analog.

In another embodiment, the CD1d-restricted antigens compriseglycolipids. In another embodiment, the CD1d-restricted antigenscomprise α-galactosylceramide, β-glucosylceramide, α-linkedglycosphingolipid from Sphingomonas spp., galactosyl diaglycerol fromBorrelia burgdorferi, lypophosphoglycan from Leishmania orphosphatidylinositol tetramannoside from Mycobacterium leprae.

In another embodiment, a tolerogenic immune response is generated in thesubject. In one embodiment, the tolerogenic immune response is thestimulation of iNKT cells. In another embodiment, the tolerogenic immuneresponse is the production of IL-4 by iNKT cells. In another embodiment,the tolerogenic immune response is the production of IL-10 by iNKTcells. In another embodiment, the tolerogenic immune response is iNKTcell stimulation. In another embodiment, the tolerogenic immune responseis iNKT cell anergy or the reduction in pathogenic iNKT cells. Inanother embodiment, the tolerogenic immune response is a reduction inIFN-γ produced by iNKT cells. In another embodiment, the tolerogenicimmune response is antigen-specific.

In another embodiment, the method further comprises administering APCpresentable antigens to the subject. In one embodiment, the APCpresentable antigens are administered prior to, concomitantly with orafter the administration of the composition comprising the firstpopulation and second population of synthetic nanocarriers. In anotherembodiment, the APC presentable antigens are coupled to the first and/orsecond populations of synthetic nanocarriers. In another embodiment, theAPC presentable antigens are coupled to a third population of syntheticnanocarriers. In one embodiment, the coupling is covalent coupling. Inanother embodiment, the coupling is noncovalent coupling. In anotherembodiment, the APC presentable antigens are not coupled to syntheticnanocarriers.

In another embodiment, the APC presentable antigens comprise MHC ClassI-restricted, MHC Class II-restricted epitopes and/or B cell epitopes.In yet another embodiment, the APC presentable antigens comprisesubstantially no B cell epitopes. In one embodiment, the APC presentableantigens are or are obtained or derived from proteins, polypeptides,polysaccharides, polynucleotides or cells.

In a further embodiment, the method further comprises administering atransplantable graft.

In another embodiment, the APC presentable antigens are autoantigens,allergens, or are associated with an inflammatory disease, fatty liverdisease, an autoimmune disease, an allergy, sickle cell disease,spontaneous abortion organ or tissue rejection or graft versus hostdisease.

In another embodiment, the load of the immunosuppressants and/orCD1d-restricted antigens on average across the first and/or secondpopulation of synthetic nanocarriers is between 0.0001% and 50%. Inanother embodiment, the load of the immunosuppressants and/orCD1d-restricted antigens on average across the first and/or secondpopulation of synthetic nanocarriers is between 0.1% and 10%.

In another embodiment, the synthetic nanocarriers of the firstpopulation and/or second population and/or third population compriselipid nanoparticles, polymeric nanoparticles, metallic nanoparticles,surfactant-based emulsions, dendrimers, buckyballs, nanowires,virus-like particles or peptide or protein particles. In anotherembodiment, the synthetic nanocarriers of the first and/or second and/orthird populations comprise lipid nanoparticles. In another embodiment,the synthetic nanocarriers of the first and/or second and/or thirdpopulations comprise liposomes. In another embodiment, the syntheticnanocarriers of the first and/or second and/or third populationscomprise metallic nanoparticles. In another embodiment, the metallicnanoparticles comprise gold nanoparticles. In another embodiment, thesynthetic nanocarriers of the first and/or second and/or thirdpopulations comprise polymeric nanoparticles. In another embodiment, thepolymeric nanoparticles comprise polymer that is anon-methoxy-terminated, pluronic polymer. In another embodiment, thepolymeric nanoparticles comprise a polyester, a polyester coupled to apolyether, polyamino acid, polycarbonate, polyacetal, polyketal,polysaccharide, polyethyloxazoline or polyethyleneimine. In anotherembodiment, the polyester comprises a poly(lactic acid), poly(glycolicacid), poly(lactic-co-glycolic acid) or polycaprolactone. In anotherembodiment, the polymeric nanoparticles comprise a polyester and apolyester coupled to a polyether. In another embodiment, the polyethercomprises polyethylene glycol or polypropylene glycol.

In another embodiment, the mean of a particle size distribution obtainedusing dynamic light scattering of the synthetic nanocarriers of thefirst and/or second and/or third population is a diameter greater than100 nm. In another embodiment, the diameter is greater than 150 nm. Inanother embodiment, the diameter is greater than 200 nm. In anotherembodiment, the diameter is greater than 250 nm. In another embodiment,the diameter is greater than 300 nm.

In another embodiment, the aspect ratio of the synthetic nanocarriers ofthe first population and/or second population and/or third population isgreater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.

In another embodiment, one or more maintenance doses of the compositioncomprising the first population and second population of syntheticnanocarriers and/or the APC presentable antigens are administered to thesubject.

In another embodiment, the method further comprises assessing thegeneration of a/the tolerogenic immune response in the subject prior toand/or after the administration of the composition comprising the firstpopulation and second population of synthetic nanocarriers and/or theAPC presentable antigens. In one embodiment, the tolerogenic immuneresponse is the stimulation of iNKT cells. In another embodiment, thetolerogenic immune response is the production of IL-4 by iNKT cells. Inanother embodiment, the tolerogenic immune response is the production ofIL-10 by iNKT cells. In another embodiment, the tolerogenic immuneresponse is iNKT cell stimulation. In another embodiment, thetolerogenic immune response is iNKT cell anergy or the reduction inpathogenic iNKT cells. In another embodiment, the tolerogenic immuneresponse is a reduction in IFN-γ produced by iNKT cells. In anotherembodiment, the tolerogenic immune response is antigen-specific.

In another embodiment, the subject has or is at risk of having aninflammatory disease, fatty liver disease, an autoimmune disease, sicklecell disease, an allergy, spontaneous abortion, organ or tissuerejection or graft versus host disease. In another embodiment, thesubject has undergone or will undergo transplantation.

In another embodiment, the administering of the synthetic nanocarriersand/or APC presentable antigens is by intravenous, intraperitoneal,transmucosal, oral, subcutaneous, pulmonary, intranasal, intradermal orintramuscular administration. In another embodiment, the administeringof the synthetic nanocarriers and/or APC presentable antigens is byinhalation or intravenous, subcutaneous or transmucosal administration.

In another aspect, a method comprising (i) producing a first populationof synthetic nanocarriers coupled to immunosuppressants, and (ii)producing a second population of synthetic nanocarriers coupled toCD1d-restricted antigens is provided.

In one embodiment, the first population and second population are thesame population. In another embodiment, the first population and secondpopulation are different populations.

In another embodiment, the method further comprises including APCpresentable antigens with the first population and/or second populationof the synthetic nanocarriers. In one embodiment, the APC presentableantigens are coupled to the first population and/or second population ofsynthetic nanocarriers. In another embodiment, the APC presentableantigens are coupled to a third population of synthetic nanocarriers. Inanother embodiment, the coupling is covalent coupling. In anotherembodiment, the coupling is non-covalent coupling. In anotherembodiment, the APC presentable antigens are not coupled to syntheticnanocarriers.

In another embodiment, the first population and second population and/orthird population of synthetic nanocarriers that are produced are asdefined in any of the compositions or methods provided herein. Inanother embodiment, the method further comprises producing a dosage formof a composition comprising the first population and second populationand/or third population of synthetic nanocarriers produced. In anotherembodiment, the method further comprises making a composition comprisingthe first population and second population of synthetic nanocarriersand/or third population of synthetic nanocarriers or the dosage formavailable to a subject for administration. In another embodiment, themethod further comprises assessing the generation of a tolerogenicimmune response with a composition comprising the first population andsecond population of synthetic nanocarriers and/or third population ofsynthetic nanocarriers or the dosage form.

In one embodiment, the tolerogenic immune response is the stimulation ofiNKT cells. In another embodiment, the tolerogenic immune response isthe production of IL-4 by iNKT cells. In another embodiment, thetolerogenic immune response is the production of IL-10 by iNKT cells. Inanother embodiment, the tolerogenic immune response is iNKT cellstimulation. In another embodiment, the tolerogenic immune response isiNKT cell anergy or the reduction in pathogenic iNKT cells. In anotherembodiment, the tolerogenic immune response is a reduction in IFN-γproduced by iNKT cells. In another embodiment, the tolerogenic immuneresponse is antigen-specific.

In another aspect, a process for producing a composition or dosage formcomprising the steps of (i) coupling a first population of syntheticnanocarriers to immunosuppressants, and (ii) coupling a secondpopulation of synthetic nanocarriers to CD1d-restricted antigens isprovided. In one embodiment, the process comprises the steps as definedin any of the methods provided herein.

In another aspect, a composition or dosage form obtainable by any of themethods or processes provided herein is provided.

In another aspect, any of the compositions or dosage forms provided maybe for use in therapy or prophylaxis.

In another aspect, any of the compositions or dosage forms provided maybe for use in a method of generating a tolerogenic immune response in asubject, the treatment or prophylaxis of allergy, sickle cell disease,autoimmune disease, inflammatory disease, fatty liver disease,spontaneous abortion, organ or tissue rejection or graft versus hostdisease or in any of the methods provided herein.

In another aspect, a use of any of the compositions or dosage formsprovided for the manufacture of a medicament for use in a method ofgenerating a tolerogenic immune response in a subject, the treatment orprophylaxis of allergy, sickle cell disease, autoimmune disease,inflammatory disease, fatty liver disease, spontaneous abortion, organor tissue rejection or graft versus host disease or in any of themethods provided herein is provided.

In another aspect, a dosage form comprising any of the compositionsprovided herein is provided.

In another embodiment, of any of the compositions and methods providedherein, the CD1d-restricted antigen comprises substantially no B cellepitopes.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides a representative example of a flow cytometric analysisof Treg cells.

FIG. 2 shows that synthetic nanocarriers comprising immunosuppressantreduces the level of CD69 expression, a marker for T cell activation.

FIG. 3 shows the level of IFN-γ expression by iNKT cells.

FIG. 4 shows the level of IL-4 expression by iNKT cells.

FIG. 5 shows the level of IL-10 expression by iNKT cells.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes. As used in this specification and the appended claims,the singular forms “a,” “an” and “the” include plural referents unlessthe content clearly dictates otherwise. For example, reference to “apolymer” includes a mixture of two or more such molecules or a mixtureof differing molecular weights of a single polymer species, reference to“a synthetic nanocarrier” includes a mixture of two or more suchsynthetic nanocarriers or a plurality of such synthetic nanocarriers,reference to “a DNA molecule” includes a mixture of two or more such DNAmolecules or a plurality of such DNA molecules, reference to “animmunosuppressant” includes mixture of two or more such materials or aplurality of immunosuppressant molecules, and the like.

As used herein, the term “comprise” or variations thereof such as“comprises” or “comprising” are to be read to indicate the inclusion ofany recited integer (e.g. a feature, element, characteristic, property,method/process step or limitation) or group of integers (e.g. features,element, characteristics, properties, method/process steps orlimitations) but not the exclusion of any other integer or group ofintegers. Thus, as used herein, the term “comprising” is inclusive anddoes not exclude additional, unrecited integers or method/process steps.

In embodiments of any of the compositions and methods provided herein,“comprising” may be replaced with “consisting essentially of” or“consisting of”. The phrase “consisting essentially of” is used hereinto require the specified integer(s) or steps as well as those which donot materially affect the character or function of the claimedinvention. As used herein, the term “consisting” is used to indicate thepresence of the recited integer (e.g. a feature, element,characteristic, property, method/process step or limitation) or group ofintegers (e.g. features, element, characteristics, properties,method/process steps or limitations) alone.

A. INTRODUCTION

NKT cells are a heterogeneous group of T cells that can be divided intoinvariant NKT cells and non-invariant NKT cells. Invariant NKT (iNKT)cells are a population of innate T cells that are restricted by thenon-classical MHC Class I molecule, CD1d. CD1d is an antigen-presentingmolecule that binds self- and foreign lipids and glycolipids and isoften found on antigen-presenting cells, but also on non-hematopoieticcells such as hepatocytes. Upon stimulation, iNKT cells, in someembodiments, are able to enhance tolerogenic effects of the immunesystem, such as by shifting to the production of anti-inflammatorycytokines (e.g., IL-10 and IL-4). As illustrated in the Examples below,it has been found that synthetic nanocarriers that compriseimmunosuppressant and CD1d-restricted antigens are able to elicit suchtolerogenic effects. It has been shown that such synthetic nanocarrierswere effective in producing the aforementioned cytokines when contactedwith the nanocarriers. In some embodiments, the compositions providedherein can result in iNKT cell anergy where their activation andproduction of cytokines is reduced (e.g., inflammatory cytokines, suchas IFN-γ). In some embodiments, such as where iNKT cell activity ispathogenic (e.g., asthma) iNKT cell anergy or a decrease in cytokines,such as IL-4, may be beneficial. In other embodiments, the compositionsprovided herein can result in iNKT cell stimulation. With thecompositions and methods provided, it is possible to cause iNKT cells topromote tolerogenic effects, which have application in a number ofdisease contexts. The compositions provided herein are also expected tobe delivered more directly at the sites of action in cells of interest,in particular APCs, and can not only result in beneficial tolerogenicimmune responses but also a reduction in off-target effects andtoxicity. This invention is useful for promoting tolerogenic immuneresponses in a variety of subjects, such as those that have or are atrisk of having an inflammatory disease, an autoimmune disease, sicklecell disease, fatty liver disease, spontaneous abortion, an allergy,organ or tissue rejection or graft verus host disease. Such subjectsalso include those who have undergone or will undergo transplantation.

The inventors have unexpectedly and surprisingly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. In particular, the inventors haveunexpectedly discovered that it is possible to provide syntheticnanocarrier compositions, and related methods, that induce a tolerogenicimmune response. The compositions described herein include compositionsthat comprise (i) a first population of synthetic nanocarriers coupledto immunosuppressants, and (ii) a second population of syntheticnanocarriers coupled to CD1d-restricted antigens. In embodiments, thecompositions further comprise an APC presentable antigen that may or maynot be coupled to the first or second population of syntheticnanocarriers or another population of synthetic nanocarriers. Thepresence of the APC presentable antigens can result in furtherantigen-specific tolerogenic immune responses.

In another aspect, dosage forms of any of the compositions herein areprovided. Such dosage forms can be administered to a subject in needthereof (e.g., in need of tolerogenic immune responses).

In another aspect, any of the compositions provided herein isadministered to a subject. The composition may be administered in anamount effective to generate a tolerogenic immune response in thesubject. In one embodiment, a composition is administered to a subjectaccording to a protocol that was previously shown to generate atolerogenic immune response in one or more subjects.

Other antigens, such as APC presentable antigens, may also beadministered to the subject provided herein. Such antigens may beadministered to a subject prior to, concomitantly with or after theadministration of the first and second populations of syntheticnanocarriers. Such antigens may or may not be coupled to the first orsecond population of synthetic nanocarriers or another population ofsynthetic nanocarriers.

In embodiments, the compositions provided may also be administered asone or more maintenance doses to a subject. In such embodiments, thecompositions provided are administered such that the generation of anundesired immune response is reduced or a desired immune response isproduced for a certain length of time. Examples of such lengths of timeare provided elsewhere herein.

In yet another aspect, a method of (i) producing a first population ofsynthetic nanocarriers coupled to immunosuppressants, and (ii) producinga second population of synthetic nanocarriers coupled to CD1d-restrictedantigens is provided.

The invention will now be described in more detail below.

B. DEFINITIONS

“Administering” or “administration” means providing a material to asubject in a manner that is pharmacologically useful.

“Allergens” are any substances that can cause an undesired (e.g., a Type1 hypersensitive) immune response (i.e., an allergic response orreaction) in a subject. Allergens include, but are not limited to, plantallergens (e.g., pollen, ragweed allergen), insect allergens, insectsting allergens (e.g., bee sting allergens), animal allergens (e.g., petallergens, such as animal dander or cat Fel d 1 antigen), latexallergens, mold allergens, fungal allergens, cosmetic allergens, drugallergens, food allergens, dust, insect venom, viruses, bacteria, etc.Food allergens include, but are not limited to milk allergens, eggallergens, nut allergens (e.g., peanut or tree nut allergens, etc.(e.g., walnuts, cashews, etc.)), fish allergens, shellfish allergens,soy allergens, legume allergens, seed allergens and wheat allergens.Insect sting allergens include allergens that are or are associated withbee stings, wasp stings, hornet stings, yellow jacket stings, etc.Insect allergens also include house dust mite allergens (e.g., Der P1antigen) and cockroach allergens. Drug allergens include allergens thatare or are associated with antibiotics, NSAIDs, anaesthetics, etc.Pollen allergens include grass allergens, tree allergens, weedallergens, flower allergens, etc. Subjects that develop or are at riskof developing an undesired immune response to any of the allergensprovided herein may be treated with any of the compositions and methodsprovided herein. Subjects that may be treated with any of thecompositions and methods provided also include those who have or are atrisk of having an allergy to any of the allergens provided.

An “allergy” also referred to herein as an “allergic condition,” is anycondition where there is an undesired (e.g., a Type 1 hypersensitive)immune response (i.e., allergic response or reaction) to a substance.Such substances are referred to herein as allergens. Allergies orallergic conditions include, but are not limited to, allergic asthma,hay fever, hives, eczema, plant allergies, bee sting allergies, petallergies, latex allergies, mold allergies, cosmetic allergies, foodallergies, allergic rhinitis or coryza, topic allergic reactions,anaphylaxis, atopic dermatitis, hypersensitivity reactions and otherallergic conditions. The allergic reaction may be the result of animmune reaction to any allergen. In some embodiments, the allergy is afood allergy. Food allergies include, but are not limited to, milkallergies, egg allergies, nut allergies, fish allergies, shellfishallergies, soy allergies or wheat allergies.

“Amount effective” in the context of a composition or dosage form foradministration to a subject refers to an amount of the composition ordosage form that produces one or more desired immune responses in thesubject, for example, such the stimulation of iNKT cells to produce IL-4and/or IL-10. Therefore, in some embodiments, an amount effective is anyamount of a composition provided herein that produces one or more ofthese desired immune responses. This amount can be for in vitro or invivo purposes. For in vivo purposes, the amount can be one that aclinician would believe may have a clinical benefit for a subject inneed of tolerization. Such subjects include those that have or are atrisk of having an inflammatory disease, an autoimmune disease, anallergy, fatty liver disease, spontaneous abortion, organ or tissuerejection or graft versus host disease. Such subjects also include thosethat have undergone or will undergo transplantation.

Amounts effective can involve only reducing the level of an undesiredimmune response, although in some embodiments, it involves preventing anundesired immune response altogether. Amounts effective can also involvedelaying the occurrence of an undesired immune response. An amount thatis effective can also be an amount of a composition provided herein thatproduces a desired therapeutic endpoint or a desired therapeutic result.Amounts effective, preferably, result in tolerance in a subject to anantigen but in some embodiments the amounts effective simply result in atolerogenic effect or a shift to a tolerogenic phenotype. Theachievement of any of the foregoing can be monitored by routine methods.Preferably, in some embodimenst, the amounts effective herein are thosethat result in the production of IL-4 and/or IL-10 by iNKT cells.

In some embodiments of any of the compositions and methods provided, theamount effective is one in which the desired immune response persists inthe subject for at least 1 week, at least 2 weeks, at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months, at least 9 months, at least 1 year, at least 2 years,at least 5 years, or longer. In other embodiments of any of thecompositions and methods provided, the amount effective is one whichproduces a measurable desired immune response, for example, a measurableincrease or decrease in an immune response (e.g., to a specificantigen), for at least 1 week, at least 2 weeks, at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months, at least 9 months, at least 1 year, at least 2 years,at least 5 years, or longer.

Amounts effective will depend, of course, on the particular subjectbeing treated; the severity of a condition, disease or disorder; theindividual patient parameters including age, physical condition, sizeand weight; the duration of the treatment; the nature of concurrenttherapy (if any); the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is generallypreferred that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reason.

In general, doses of the immunosuppressants and/or antigens in thecompositions of the invention can range from about 10 μg/kg to about100,000 μg/kg. In some embodiments, the doses can range from about 0.1mg/kg to about 100 mg/kg. In still other embodiments, the doses canrange from about 0.1 mg/kg to about 25 mg/kg, about 25 mg/kg to about 50mg/kg, about 50 mg/kg to about 75 mg/kg or about 75 mg/kg to about 100mg/kg. Alternatively, the dose can be administered based on the numberof synthetic nanocarriers that provide the desired amount ofimmunosuppressants and/or antigens. For example, useful doses includegreater than 10⁶, 10⁷, 10⁸, 10⁹ or 10¹⁰ synthetic nanocarriers per dose.Other examples of useful doses include from about 1×10⁶ to about 1×10¹⁰,about 1×10⁷ to about 1×10⁹ or about 1×10⁸ to about 1×10⁹ syntheticnanocarriers per dose.

“Antigen” means a CD1d-restricted antigen, B cell antigen or T cellantigen. “Type(s) of antigens” means molecules that share the same, orsubstantially the same, antigenic characteristics. In some embodiments,antigens may be lipids, proteins, polypeptides, peptides, lipoproteins,glycolipids, polynucleotides, polysaccharides or are contained orexpressed in cells. In some embodiments, such as when the antigens arenot well defined or characterized, the antigens may be contained withina cell or tissue preparation, cell debris, cell exosomes, conditionedmedia, etc. An antigen can be combined with the synthetic nanocarriersin the same form as what a subject is exposed to that causes anundesired immune response but may also be a fragment or derivativethereof. When a fragment or derivative, however, a desired immuneresponse to the form encountered by such a subject is the preferableresult with the compositions and methods provided.

“Antigen-specific” refers to any immune response that results from thepresence of the antigen, or portion thereof, or that generates moleculesthat specifically recognize or bind the antigen.

“Antigens associated” with a disease, disorder or condition providedherein are antigens that can generate an undesired immune responseagainst, as a result of, or in conjunction with the disease, disorder orcondition; the cause of the disease, disorder or condition (or a symptomor effect thereof); and/or can generate an undesired immune responsethat is a symptom, result or effect of the disease, disorder orcondition. Preferably, in some embodiments, the use of an antigenassociated with a disease, disorder or condition, etc. in thecompositions and methods provided herein will lead to a tolerogenicimmune response against the antigen and/or the cells, by, on or in whichthe antigen is expressed. The antigens can be in the same form asexpressed in a subject with the disease, disorder or condition but mayalso be a fragment or derivative thereof. When a fragment or derivative,however, a desired immune response to the form expressed in such asubject is the preferable result with the compositions and methodsprovided.

In one embodiment, the antigen is an antigen associated with aninflammatory disease, autoimmune disease, organ or tissue rejection orgraft versus host disease. Such antigens include autoantigens, such asmyelin basic protein, collagen (e.g., collagen type 11), human cartilagegp 39, chromogranin A, gp130-RAPS, proteolipid protein, fibrillarin,nuclear proteins, nucleolar proteins (e.g., small nucleolar protein),thyroid stimulating factor receptor, histones, glycoprotein gp 70,ribosomal proteins, pyruvate dehydrogenase dehydrolipoamideacetyltransferase, hair follicle antigens, human tropomyosin isoform 5,mitochondrial proteins, pancreatic β-cell proteins, myelinoligodendrocyte glycoprotein, insulin, glutamic acid decarboxylase(GAD), gluten, and fragments or derivatives thereof. Other autoantigensare provided in Table 1 below.

Antigens also include those associated with organ or tissue rejection.Examples of such antigens include, but are not limited to, antigens fromallogeneic cells, e.g., antigens from an allogeneic cell extract andantigens from other cells, such as endothelial cell antigens.

Antigens also include those associated with an allergy. Such antigensinclude the allergens described elsewhere herein.

Antigens also include those associated with a transplantable graft. Suchantigens are associated with (specific for) a transplantable graft, oran undesired immune response in a recipient of a transplantable graftthat is generated as a result of the introduction of the transplantablegraft in the recipient, that can be presented for recognition by cellsof the immune system and that can generate an undesired immune response.Transplant antigens include those associated with organ or tissuerejection or graft versus host disease. Transplant antigens may beobtained or derived from cells of a biological material or frominformation related to a transplantable graft. Transplant antigensgenerally include lipids, proteins, polypeptides, peptides,lipoproteins, glycolipids, lipids, polynucleotides or are contained orexpressed in cells. Information related to a transplantable graft is anyinformation about a transplantable graft that can be used to obtain orderive transplant antigens. Such information includes information aboutantigens that would be expected to be present in or on cells of atransplantable graft such as, for example, sequence information, typesor classes of antigens and/or their presentation restrictions. Suchinformation may also include information about the type oftransplantable graft (e.g, autograft, allograft, xenograft), themolecular and cellular composition of the graft, the bodily locationfrom which the graft is derived or to which the graft is to betransplanted (e.g., whole or partial organ, skin, bone, nerves, tendon,neurons, blood vessels, fat, cornea, etc.).

“APC presentable antigen” means an antigen that can be presented forrecognition by cells of the immune system, such as presented by antigenpresenting cells, including but not limited to dendritic cells, B cellsor macrophages. The APC presentable antigen can be presented forrecognition by, for example, T cells. Such antigens may be recognized byand trigger an immune response in a T cell via presentation of theantigen or portion thereof bound to a Class I or Class II majorhistocompatability complex molecule (MHC), or bound to a CD1d molecule.CD1d is an antigen-presenting molecule that binds self and foreignlipids and glycolipids, and is often found on antigen presenting cells.It is also found on non-hematopoietic cells such as hepatocytes. CD1dcontains a hydrophobic groove which binds hydrophobic lipids, usuallyfor presentation to iNKT cells. Preferably, one or more tolerogenicimmune responses specific to the APC presentable antigen results withthe compositions provided herein.

“Assessing an immune response” refers to any measurement ordetermination of the level, presence or absence, reduction, increase in,etc. of an immune response in vitro or in vivo. Such measurements ordeterminations may be performed on one or more samples obtained from asubject. Such assessing can be performed with any of the methodsprovided herein or otherwise known in the art.

An “at risk” subject is one in which a health practitioner believes hasa chance of having a disease, disorder or condition as provided hereinor is one a health practitioner believes has a chance of experiencing anundesired immune response as provided herein.

An “autoimmune disease” is any disease where the immune system mounts anundesired immune response against self (e.g., one or more autoantigens).In some embodiments, an autoimmune disease comprises an aberrantdestruction of cells of the body as part of the self-targeted immuneresponse. In some embodiments, the destruction of self manifests in themalfunction of an organ, for example, the colon or pancreas. Examples ofautoimmune diseases are described elsewhere herein. Additionalautoimmune diseases will be known to those of skill in the art and theinvention is not limited in this respect.

“Average”, as used herein, refers to the arithmetic mean unlessotherwise noted.

“B cell antigen” means any antigen that triggers an immune response in aB cell (e.g., an antigen that is specifically recognized by a B cell ora receptor thereon). In some embodiments, an antigen that is a T cellantigen is also a B cell antigen. In other embodiments, the T cellantigen is not also a B cell antigen. B cell antigens include, but arenot limited to proteins, peptides, small molecules, and carbohydrates.In some embodiments, the B cell antigen comprises a non-protein antigen(i.e., not a protein or peptide antigen). In some embodiments, the Bcell antigen comprises a autoantigen. In other embodiments, the B cellantigen is obtained or derived from an allergen, autoantigen,therapeutic protein, or transplantable graft.

“CD1d-restricted antigen” means an antigen that binds to, is presentedby or forms a CD1 complex. CD1d is an MHC-like molecule with ahydrophobic binding groove which binds lipid (e.g., hydrophobic)antigens. The CD1d-restricted antigen can be presented for recognitionby iNKT cells in order to activate them to enhance tolerogenic immuneresponses, in some embodiments, in the context of the compositions ofthe invention. Generally, CD1d-restricted antigens are lipids presentedto invariant NKT cells. Examples of CD1d-restricted antigens includelipids and glycolipids and portions thereof. Specific examples includemicrobial lipids such as fatty acids, phospholipids, diacyglycerol,sphingolipids, glycolipids and lipopeptides. Other examples ofCD1d-restricted antigens include Mycobacterium tuberculosis antigens,such as fatty acids (e.g., mycolic acids), glycosylated mycolic acids(e.g., GMM, glucose monomycolate), sulfated acyl trehalose (sulfolipid,diacylated sulfoglycolipid), lipopeptide dideoxymycobactin (DDM),isoprenoid based lipids (e.g., MPM, mannosyl-b1-phosphomycoketide),glycerol based lipids (e.g., PIM's, phosphatidylinositol mannosides) andhyperglycosylated form of PIMs, mannosylated lipoarabinomannan(Man-LAM), and mannosyl-b1-phosphoheptaprenol (MPP); Sphingomonas spp.antigen, such as α-glucoronsylceramide (GSL-1); B. burgdorferi antigen,such as α-galactosyldiacylglycerol (GalDAG); L. donovani antigen, suchas lipophosphoglycan (LPG); mammalian/self-antigens, such asphosphatidylinositol (PI), phosphatidylglycerol (PG),phosphatidylethanolamine (PE), GM1 (a ganglioside), GD3 (a ganglioside),β-glucosylceramide, sulfatide, and isoglobotrihexosylceramide (iGB3);synthetic/sponge antigen, such as α-galactosylceramide (alpha-GC) or ananalog thereof; β-glucosylceramide, α-linked glycosphingolipids (fromSphingomonas spp.), galactosyl diacylglycerols (from Borreliaburgdorferi), lypophosphoglycan (from Leishmania donovani) andphosphatidylinositol tetramannoside (PIM4) (from Mycobacterium leprae),etc. CD1d-restricted antigens can, in some embodiments, be associatedwith a disease, disorder or condition as provided herein. For additionallipids and/or glycolipids useful as a CD1d-restricted antigens, see V.Cerundolo et al., “Harnessing invariant NKT cells in vaccinationstrategies.” Nature Rev Immun, 9:28-38 (2009).

“Concomitantly” means administering two or more substances to a subjectin a manner that is correlated in time, preferably sufficientlycorrelated in time so as to provide a modulation in an immune response.In embodiments, concomitant administration may occur throughadministration of two or more substances in the same dosage form. Inother embodiments, concomitant administration may encompassadministration of two or more substances in different dosage forms, butwithin a specified period of time, preferably within 1 month, morepreferably within 1 week, still more preferably within 1 day, and evenmore preferably within 1 hour.

“Couple” or “Coupled” or “Couples” (and the like) means to chemicallyassociate one entity (for example a moiety) with another. In someembodiments, the coupling is covalent, meaning that the coupling occursin the context of the presence of a covalent bond between the twoentities. In non-covalent embodiments, the non-covalent coupling ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,encapsulation is a form of coupling.

“Derived” means prepared from a material or information related to amaterial but is not “obtained” from the material. Such materials may besubstantially modified or processed forms of materials taken directlyfrom a biological material. Such materials also include materialsproduced from information related to a biological material.

“Dosage form” means a pharmacologically and/or immunologically activematerial in a medium, carrier, vehicle, or device suitable foradministration to a subject.

“Encapsulate” means to enclose at least a portion of a substance withina synthetic nanocarrier. In some embodiments, a substance is enclosedcompletely within a synthetic nanocarrier. In other embodiments, most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. In other embodiments,no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed tothe local environment. Encapsulation is distinct from absorption, whichplaces most or all of a substance on a surface of a syntheticnanocarrier, and leaves the substance exposed to the local environmentexternal to the synthetic nanocarrier.

“Epitope”, also known as an antigenic determinant, is the part of anantigen that is recognized by the immune system, specifically by, forexample, iNKT cells, antibodies, B cells, or T cells. In someembodiments, the epitope itself is an antigen.

A number of epitopes are known to those of skill in the art, andexemplary epitopes suitable according to some aspects of this inventioninclude, but are not limited to those listed in the Immune EpitopeDatabase (www.immuneepitope.org, Vita R, Zarebski L, Greenbaum J A,Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters B. The immuneepitope database 2.0. Nucleic Acids Res. 2010 January; 38(Databaseissue):D854-62; the entire contents of which as well as all databaseentries of IEDB version 2.4, August 2011, and particularly all epitopesdisclosed therein, are incorporated herein by reference). Epitopes canalso be identified with publicly available algorithms, for example, thealgorithms described in Wang P, Sidney J, Kim Y, Sette A, Lund O,Nielsen M, Peters B. 2010. peptide binding predictions for HLA DR, DPand DQ molecules. BMC Bioinformatics 2010, 11:568; Wang P, Sidney J, DowC, Mothé B, Sette A, Peters B. 2008. A systematic assessment of MHCclass II peptide binding predictions and evaluation of a consensusapproach. PLoS Comput Biol. 4(4):e1000048; Nielsen M, Lund 0.2009.NN-align. An artificial neural network-based alignment algorithm for MHCclass II peptide binding prediction. BMC Bioinformatics. 10:296; NielsenM, Lundegaard C, Lund O. 2007. Prediction of MHC class II bindingaffinity using SMM-align, a novel stabilization matrix alignment method.BMC Bioinformatics. 8:238; Bui H H, Sidney J, Peters B, Sathiamurthy M,Sinichi A, Purton K A, Motile B R, Chisari F V, Watkins D I, Sette A.2005. Immunogenetics. 57:304-314; Sturniolo T, Bono E, Ding J,Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M, Gallazzi F, Protti MP, Sinigaglia F, Hammer J. 1999. Generation of tissue-specific andpromiscuous HLA ligand databases using DNA microarrays and virtual HLAclass II matrices. Nat. Biotechnol. 17(6):555-561; Nielsen M, LundegaardC, Worning P, Lauemoller S L, Lamberth K, Buus S, Brunak S, Lund O.2003. Reliable prediction of T-cell epitopes using neural networks withnovel sequence representations. Protein Sci 12:1007-1017; Bui H H,Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton K A, Mothe B R,Chisari F V, Watkins D I, Sette A. 2005. Automated generation andevaluation of specific MHC binding predictive tools: ARB matrixapplications. Immunogenetics 57:304-314; Peters B, Sette A. 2005.Generating quantitative models describing the sequence specificity ofbiological processes with the stabilized matrix method. BMCBioinformatics 6:132; Chou P Y, Fasman G D. 1978. Prediction of thesecondary structure of proteins from their amino acid sequence. AdvEnzymol Relat Areas Mol Biol 47:45-148; Emini E A, Hughes J V, Perlow DS, Boger J. 1985. Induction of hepatitis A virus-neutralizing antibodyby a virus-specific synthetic peptide. J Virol 55:836-839; Karplus P A,Schulz G E. 1985. Prediction of chain flexibility in proteins.Naturwissenschaften 72:212-213; Kolaskar A S, Tongaonkar P C. 1990. Asemi-empirical method for prediction of antigenic determinants onprotein antigens. FEBS Lett276:172-174; Parker J M, Guo D, Hodges R S.1986. New hydrophilicity scale derived from high-performance liquidchromatography peptide retention data: correlation of predicted surfaceresidues with antigenicity and X-ray-derived accessible sites.Biochemistry 25:5425-5432; Larsen J E, Lund O, Nielsen M. 2006. Improvedmethod for predicting linear B-cell epitopes. Immunome Res 2:2;Ponomarenko J V, Bourne P E. 2007. Antibody-protein interactions:benchmark datasets and prediction tools evaluation. BMC Struct Biol7:64; Haste Andersen P, Nielsen M, Lund 0.2006. Prediction of residuesin discontinuous B-cell epitopes using protein 3D structures. ProteinSci 15:2558-2567; Ponomarenko J V, Bui H, Li W, Fusseder N, Bourne P E,Sette A, Peters B. 2008. ElliPro: a new structure-based tool for theprediction of antibody epitopes. BMC Bioinformatics 9:514; Nielsen M,Lundegaard C, Blicher T, Peters B, Sette A, Justesen S, Buus S, and Lund0.2008. PLoS Comput Biol.4(7)e1000107. Quantitative predictions ofpeptide binding to any HLA-DR molecule of known sequence: NetMHCIIpan;the entire contents of each of which are incorporated herein byreference for disclosure of methods and algorithms for theidentification of epitopes.

“Generating” means causing an action, such as an immune response (e.g.,a tolerogenic immune response) to occur, either directly oneself orindirectly, such as, but not limited to, an unrelated third party thattakes an action through reliance on one's words or deeds.

“Identifying” is any action or set of actions that allows a clinician torecognize a subject as one who may benefit from the methods andcompositions provided herein. Preferably, the identified subject is onewho is in need of a tolerogenic immune response as provided herein. Theaction or set of actions may be either directly oneself or indirectly,such as, but not limited to, an unrelated third party that takes anaction through reliance on one's words or deeds.

“Immunosuppressant” means a compound that causes an APC to have animmunosuppressive (e.g., tolerogenic effect). An immunosuppressiveeffect generally refers to the production or expression of cytokines orother factors by the APC that reduces, inhibits or prevents an undesiredimmune response or that promotes a desired immune response. When the APCresults in an immunosuppressive effect on immune cells that recognize anantigen presented by the APC, the immunosuppressive effect is said to bespecific to the presented antigen. Such effect is also referred toherein as a tolerogenic effect. Without being bound by any particulartheory, it is thought that the immunosuppressive or tolerogenic effectis a result of the immunosuppressant being delivered to the APC,preferably in the presence of an antigen, such as an epitope, (e.g., anadministered antigen or one that is already present in vivo).Accordingly, the immunosuppressant includes compounds that provide atolerogenic immune response to an antigen that may or may not beprovided in the same composition or a different composition. In oneembodiment, the immunosuppressant is one that causes an APC to promote aregulatory phenotype in one or more immune effector cells. For example,the regulatory phenotype may be characterized by the production,induction, stimulation or recruitment of iNKT cells, e.g., such cellsthat produce IL-4 and/or IL-10. As another example, the regulatoryphenotype can be characterized in the inhibition of the production,induction, stimulation or recruitment of iNKT cells that produce IFN-γ.As another example, the phenotype can be characterized by iNKT cellstimulation, while in other embodiment, it is characterized by iNKT cellanergy or the reduction of pathogenic iNKT cells. The regulatoryphenotype, in some embodiments, may be the induction of FoxP3 in iNKTcells. In one embodiment, the immunosuppressant is one that affects theresponse of the APC after it processes an antigen. In anotherembodiment, the immunosuppressant is not one that interferes with theprocessing of the antigen. In a further embodiment, theimmunosuppressant is not an apoptotic-signaling molecule. In anotherembodiment, the immunosuppressant is not a phospholipid.

Immunosuppressants include, but are not limited to, statins; mTORinhibitors, such as rapamycin or a rapamycin analog; TGF-β signalingagents; TGF-β receptor agonists; histone deacetylase inhibitors, such asTrichostatin A; corticosteroids; inhibitors of mitochondrial function,such as rotenone; P38 inhibitors; NF-κβ inhibitors, such as 6Bio,Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists;prostaglandin E2 agonists (PGE2), such as Misoprostol; phosphodiesteraseinhibitors, such as phosphodiesterase 4 inhibitor (PDE4), such asRolipram; proteasome inhibitors; kinase inhibitors; G-protein coupledreceptor agonists; G-protein coupled receptor antagonists;glucocorticoids; retinoids; cytokine inhibitors; cytokine receptorinhibitors; cytokine receptor activators; peroxisomeproliferator-activated receptor antagonists; peroxisomeproliferator-activated receptor agonists; histone deacetylaseinhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KBinhibitors, such as TGX-221; autophagy inhibitors, such as3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasomeinhibitor I (PSI); and oxidized ATPs, such as P2X receptor blockers.Immunosuppressants also include IDO, vitamin D3, cyclosporins, such ascyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol,azathiopurine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG),FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirinand other COX inhibitors, niflumic acid, estriol and triptolide. Inembodiments, the immunosuppressant may comprise any of theimmunosuppressive agents provided herein.

The immunosuppressant can be a compound that directly provides theimmunosuppressive (e.g., tolerogenic) effect on APCs or it can be acompound that provides the immunosuppressive (e.g., tolerogenic) effectindirectly (i.e., after being processed in some way afteradministration). Immunosuppressants, therefore, include prodrug forms ofany of the compounds provided herein.

Immunosuppressants also include nucleic acids that encode the peptides,polypeptides or proteins provided herein that result in animmunosuppressive (e.g., tolerogenic) immune response. In embodiments,therefore, the immunosuppressant is a nucleic acid that encodes apeptide, polypeptide or protein that results in an immunosuppressive(e.g., tolerogenic) immune response, and it is the nucleic acid that iscoupled to the synthetic nanocarrier.

The nucleic acid may be DNA or RNA, such as mRNA. In embodiments, theinventive compositions comprise a complement, such as a full-lengthcomplement, or a degenerate (due to degeneracy of the genetic code) ofany of the nucleic acids provided herein. In embodiments, the nucleicacid is an expression vector that can be transcribed when transfectedinto a cell line. In embodiments, the expression vector may comprise aplasmid, retrovirus, or an adenovirus amongst others. Nucleic acids canbe isolated or synthesized using standard molecular biology approaches,for example by using a polymerase chain reaction to produce a nucleicacid fragment, which is then purified and cloned into an expressionvector. Additional techniques useful in the practice of this inventionmay be found in Current Protocols in Molecular Biology 2007 by JohnWiley and Sons, Inc.; Molecular Cloning: A Laboratory Manual (ThirdEdition) Joseph Sambrook, Peter MacCallum Cancer Institute, Melbourne,Australia; David Russell, University of Texas Southwestern MedicalCenter, Dallas, Cold Spring Harbor.

In embodiments, the immunosuppressants provided herein are coupled tosynthetic nanocarriers. In preferable embodiments, the immunosuppressantis an element that is in addition to the material that makes up thestructure of the synthetic nanocarrier. For example, in one embodiment,where the synthetic nanocarrier is made up of one or more polymers, theimmunosuppressant is a compound that is in addition and coupled to theone or more polymers. As another example, in one embodiment, where thesynthetic nanocarrier is made up of one or more lipids, theimmunosuppressant is again in addition and coupled to the one or morelipids. In embodiments, such as where the material of the syntheticnanocarrier also results in an immunosuppressive (e.g., tolerogenic)effect, the immunosuppressant is an element present in addition to thematerial of the synthetic nanocarrier that results in animmunosuppressive (e.g., tolerogenic) effect.

Other exemplary immunosuppressants include, but are not limited, smallmolecule drugs, natural products, antibodies (e.g., antibodies againstCD20, CD3, CD4), biologics-based drugs, carbohydrate-based drugs,nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers,methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3;tacrolimus (FK506), etc. Further immunosuppressants, are known to thoseof skill in the art, and the invention is not limited in this respect.

“Inflammatory disease” means any disease, disorder or condition in whichundesired inflammation occurs.

“Load” of the immunosuppressant or antigen is the amount of theimmunosuppressant or antigen coupled to a synthetic nanocarrier based onthe total weight of materials in an entire synthetic nanocarrier(weight/weight). Generally, the load is calculated as an average acrossa population of synthetic nanocarriers. In one embodiment, the load ofthe immunosuppressant on average across the first population ofsynthetic nanocarriers is between 0.0001% and 50%. In anotherembodiment, the load of the antigen on average across the first and/orsecond population of synthetic nanocarriers is between 0.0001% and 50%.In yet another embodiment, the load of the immunosuppressant and/orantigen is between 0.01% and 20%. In a further embodiment, the load ofthe immunosuppressant and/or antigen is between 0.1% and 10%. In still afurther embodiment, the load of the immunosuppressant and/or antigen isbetween 1% and 10%. In yet another embodiment, the load of theimmunosuppressant and/or the antigen is at least 0.1%, at least 0.2%, atleast 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%,at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, at least 13%, at least14%, at least 15%, at least 16%, at least 17%, at least 18%, at least19% or at least 20% on average across a population of syntheticnanocarriers. In yet a further embodiment, the load of theimmunosuppressant and/or the antigen is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% on average across apopulation of synthetic nanocarriers. In some embodiments of the aboveembodiments, the load of the immunosuppressant and/or the antigen is nomore than 25% on average across a population of synthetic nanocarriers.In embodiments, the load is calculated as described in the Examples.

In embodiments of any of the compositions and methods provided herein,the load may be calculated as follows: Approximately 3 mg of syntheticnanocarriers are collected and centrifuged to separate supernatant fromsynthetic nanocarrier pellet. Acetonitrile is added to the pellet, andthe sample is sonicated and centrifuged to remove any insolublematerial. The supernatant and pellet are injected on RP-HPLC andabsorbance is read at 278 nm. The μg found in the pellet is used tocalculate % entrapped (load), μg in supernatant and pellet are used tocalculate total μg recovered.

“Maintenance dose” refers to a dose that is administered to a subject,after an initial dose has resulted in an immunosuppressive (e.g.,tolerogenic) response in a subject, to sustain a desiredimmunosuppressive (e.g., tolerogenic) response. A maintenance dose, forexample, can be one that maintains the tolerogenic effect achieved afterthe initial dose, prevents an undesired immune response in the subject,or prevents the subject becoming a subject at risk of experiencing anundesired immune response, including an undesired level of an immuneresponse. In some embodiments, the maintenance dose is one that issufficient to sustain an appropriate level of a desired immune response.In some embodiments, the maintentance dose is sufficient to sustain anappropriate level of certain iNKT cell activities or defend against achallenge with an agent that results in an undesired immune response.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cuboidal synthetic nanocarrier, theminimum dimension of a synthetic nanocarrier would be the smallest ofits height, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 100 nm. In an embodiment, a maximumdimension of at least 75%, preferably at least 80%, more preferably atleast 90%, of the synthetic nanocarriers in a sample, based on the totalnumber of synthetic nanocarriers in the sample, is equal to or less than5 μm. Preferably, a minimum dimension of at least 75%, preferably atleast 80%, more preferably at least 90%, of the synthetic nanocarriersin a sample, based on the total number of synthetic nanocarriers in thesample, is greater than 110 nm, more preferably greater than 120 nm,more preferably greater than 130 nm, and more preferably still greaterthan 150 nm. Aspects ratios of the maximum and minimum dimensions ofinventive synthetic nanocarriers may vary depending on the embodiment.For instance, aspect ratios of the maximum to minimum dimensions of thesynthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferablyfrom 1:1 to 1000:1, still more preferably from 1:1 to 100:1, and yetmore preferably from 1:1 to 10:1. Preferably, a maximum dimension of atleast 75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample is equal to or less than 3 μm, morepreferably equal to or less than 2 μm, more preferably equal to or lessthan 1 μm, more preferably equal to or less than 800 nm, more preferablyequal to or less than 600 nm, and more preferably still equal to or lessthan 500 nm. In preferred embodiments, a minimum dimension of at least75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or greater than 100nm, more preferably equal to or greater than 120 nm, more preferablyequal to or greater than 130 nm, more preferably equal to or greaterthan 140 nm, and more preferably still equal to or greater than 150 nm.Measurement of synthetic nanocarrier dimensions (e.g., diameter) isobtained by suspending the synthetic nanocarriers in a liquid (usuallyaqueous) media and using dynamic light scattering (DLS) (e.g. using aBrookhaven ZetaPALS instrument). For example, a suspension of syntheticnanocarriers can be diluted from an aqueous buffer into purified waterto achieve a final synthetic nanocarrier suspension concentration ofapproximately 0.01 to 0.1 mg/mL. The diluted suspension may be prepareddirectly inside, or transferred to, a suitable cuvette for DLS analysis.The cuvette may then be placed in the DLS, allowed to equilibrate to thecontrolled temperature, and then scanned for sufficient time to acquirea stable and reproducible distribution based on appropriate inputs forviscosity of the medium and refractive indicies of the sample. Theeffective diameter, or mean of the distribution, is then reported.“Dimension” or “size” or “diameter” of synthetic nanocarriers means themean of a particle size distribution obtained using dynamic lightscattering.

“MHC” refers to major histocompatibility complex, a large genomic regionor gene family found in most vertebrates that encodes MHC molecules thatdisplay fragments or epitopes of processed proteins on the cell surface.The presentation of MHC:peptide on cell surfaces allows for surveillanceby immune cells, usually a T cell. There are two general classes of MHCmolecules: Class I and Class II. Generally, Class I MHC molecules arefound on nucleated cells and present peptides to cytotoxic T cells.Class II MHC molecules are found on certain immune cells, chieflymacrophages, B cells and dendritic cells, collectively known asprofessional APCs. The best-known genes in the MHC region are the subsetthat encodes antigen-presenting proteins on the cell surface. In humans,these genes are referred to as human leukocyte antigen (HLA) genes.

“Non-methoxy-terminated polymer” means a polymer that has at least oneterminus that ends with a moiety other than methoxy. In someembodiments, the polymer has at least two termini that ends with amoiety other than methoxy. In other embodiments, the polymer has notermini that ends with methoxy. “Non-methoxy-terminated, pluronicpolymer” means a polymer other than a linear pluronic polymer withmethoxy at both termini. Polymeric nanoparticles as provided herein cancomprise non-methoxy-terminated polymers or non-methoxy-terminated,pluronic polymers.

“Obtained” means taken directly from a material and used withsubstantially no modification and/or processing.

“Pharmaceutically acceptable excipient” means a pharmacologicallyinactive material used together with the recited synthetic nanocarriersto formulate the inventive compositions. Pharmaceutically acceptableexcipients comprise a variety of materials known in the art, includingbut not limited to saccharides (such as glucose, lactose, and the like),preservatives such as antimicrobial agents, reconstitution aids,colorants, saline (such as phosphate buffered saline), and buffers.

“Protocol” refers to any dosing regimen of one or more substances to asubject. A dosing regimen may include the amount, frequency and/or modeof administration. In some embodiments, such a protocol may be used toadminister one or more compositions of the invention to one or more testsubjects. Immune responses in these test subject can then be assessed todetermine whether or not the protocol was effective in reducing anundesired immune response or generating a desired immune response (e.g.,the promotion of a tolerogenic effect). Any other therapeutic and/orprophylactic effect may also be assessed instead of or in addition tothe aforementioned immune responses. Whether or not a protocol had adesired effect can be determined using any of the methods providedherein or otherwise known in the art. For example, a population of cellsmay be obtained from a subject to which a composition provided hereinhas been administered according to a specific protocol in order todetermine whether or not specific immune cells, cytokines, antibodies,etc. were reduced, generated, activated, etc. Useful methods fordetecting the presence and/or number of immune cells include, but arenot limited to, flow cytometric methods (e.g., FACS) andimmunohistochemistry methods. Antibodies and other binding agents forspecific staining of immune cell markers, are commercially available.Such kits typically include staining reagents for multiple antigens thatallow for FACS-based detection, separation and/or quantitation of adesired cell population from a heterogeneous population of cells.

“Providing a subject” is any action or set of actions that causes aclinician to come in contact with a subject and administer a compositionprovided herein thereto or to perform a method provided hereinthereupon. Preferably, the subject is one who is in need of atolerogenic immune response as provided herein. The action or set ofactions may be either directly oneself or indirectly, such as, but notlimited to, an unrelated third party that takes an action throughreliance on one's words or deeds.

“Subject” means animals, including warm blooded mammals such as humansand primates; avians; domestic household or farm animals such as cats,dogs, sheep, goats, cattle, horses and pigs; laboratory animals such asmice, rats and guinea pigs; fish; reptiles; zoo and wild animals; andthe like.

“Substantially no B cell epitopes” refers to the absence of B cellepitopes in an amount (by itself, within the context of the antigen, inconjunction with a carrier or in conjunction with an inventivecomposition) that stimulates substantial activation of a B cellresponse. In embodiments, a composition with substantially no B cellepitopes does not contain a measurable amount of B cell epitopes of anantigen. In other embodiments, such a composition may comprise ameasurable amount of B cell epitopes of an antigen but said amount isnot effective to generate a measurable B cell immune response (byitself, within the context of the antigen, in conjunction with a carrieror in conjunction with an inventive composition), such asantigen-specific antibody production or antigen-specific B cellproliferation and/or activity, or is not effective to generate asignificant measurable B cell immune response (by itself, within thecontext of the antigen, in conjunction with a carrier or in conjunctionwith an inventive composition). In some embodiments, a significantmeasurable B cell immune response is one that produces or would beexpected to produce an adverse clinical result in a subject. In otherembodiments, a significant measurable B cell immune response is one thatis greater than the level of the same type of immune response (e.g.,antigen-specific antibody production or antigen-specific B cellproliferation and/or activity) produced by a control antigen (e.g., oneknown not to comprise B cell epitopes of the antigen or to stimulate Bcell immune responses). In some embodiments, a significant measurable Bcell immune response, such as a measurement of antibody titers (e.g., byELISA) is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 15-fold, 20-fold or more greater than the same type ofresponse produced by a control (e.g., control antigen). In otherembodiments, a composition with substantially no B cell epitopes is onethat produces little to no antigen-specific antibody titers (by itself,within the context of the antigen, in conjunction with a carrier or inconjunction with an inventive composition). Such compositions includethose that produce an antibody titer (as an EC50 value) of less than500, 400, 300, 200, 100, 50, 40, 30, 20 or 10. In other embodiments, asignificant measurable B cell immune response, is a measurement of thenumber or proliferation of B cells that is 10%, 25%, 50%, 100%, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,15-fold, 20-fold or more greater that the same type of response producedby a control. Other methods for measuring B cell responses are known tothose of ordinary skill in the art.

In embodiments, to ensure that a composition comprises substantially noB cell epitopes, antigens are selected such that they do not comprise Bcell epitopes for coupling to the synthetic nanocarriers as providedherein. In other embodiments, to ensure that a composition comprisessubstantially no B cell epitopes of an antigen, the syntheticnanocarriers coupled to the antigen are produced and tested for B cellimmune responses (e.g., antigen-specific antibody production, B cellproliferation and/or activity). Compositions that exhibit the desiredproperties may then be selected.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are generally includedas synthetic nanocarriers, however in certain embodiments the syntheticnanocarriers do not comprise albumin nanoparticles. In embodiments,inventive synthetic nanocarriers do not comprise chitosan. In otherembodiments, inventive synthetic nanocarriers are not lipid-basednanoparticles. In further embodiments, inventive synthetic nanocarriersdo not comprise a phospholipid.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles (also referred to herein as lipidnanoparticles, i.e., nanoparticles where the majority of the materialthat makes up their structure are lipids), polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles (i.e., particles that areprimarily made up of viral structural proteins but that are notinfectious or have low infectivity), peptide or protein-based particles(also referred to herein as protein particles, i.e., particles where themajority of the material that makes up their structure are peptides orproteins) (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cuboidal, pyramidal,oblong, cylindrical, toroidal, and the like. Synthetic nanocarriersaccording to the invention comprise one or more surfaces. Exemplarysynthetic nanocarriers that can be adapted for use in the practice ofthe present invention comprise: (1) the biodegradable nanoparticlesdisclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymericnanoparticles of Published US Patent Application 20060002852 to Saltzmanet al., (3) the lithographically constructed nanoparticles of PublishedUS Patent Application 20090028910 to DeSimone et al., (4) the disclosureof WO 2009/051837 to von Andrian et al., (5) the nanoparticles disclosedin Published US Patent Application 2008/0145441 to Penades et al., (6)the protein nanoparticles disclosed in Published US Patent Application20090226525 to de los Rios et al., (7) the virus-like particlesdisclosed in published US Patent Application 20060222652 to Sebbel etal., (8) the nucleic acid coupled virus-like particles disclosed inpublished US Patent Application 20060251677 to Bachmann et al., (9) thevirus-like particles disclosed in WO2010047839A1 or WO2009106999A2, (10)the nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010), or (11) apoptotic cells, apoptotic bodies or the synthetic orsemisynthetic mimics disclosed in U.S. Publication 2002/0086049. Inembodiments, synthetic nanocarriers may possess an aspect ratio greaterthan 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersexclude virus-like particles. In embodiments, synthetic nanocarriers maypossess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5,1:7, or greater than 1:10.

“T cell antigen” means a CD4+ T-cell antigen, CD8+ cell antigen or a CD1d-restricted antigen. “CD4+ T-cell antigen” means any antigen that isrecognized by and triggers an immune response in a CD4+ T-cell e.g., anantigen that is specifically recognized by a T-cell receptor on a CD4+ Tcell via presentation of the antigen or portion thereof bound to a ClassII major histocompatability complex molecule (MHC). “CD8+ T cellantigen” means any antigen that is recognized by and triggers an immuneresponse in a CD8+ T-cell e.g., an antigen that is specificallyrecognized by a T-cell receptor on a CD8+ T cell via presentation of theantigen or portion thereof bound to a Class I major histocompatabilitycomplex molecule (MHC). “CD1d-restricted antigen” means an antigen thatcomprises one or more epitopes, or portions, that bind to, complex to orare presented by CD1d molecules. Generally, CD1d-restricted T cellantigens are lipids presented to invariant NKT cells. CD1d-restricted Tcell antigens may comprise one or more lipids, or glycolipids, includingbut not limited to: α-galactosylceramide (α-GalCer), α-linkedglycosphingolipids (from Sphingomonas spp.), galactosyl diacylglycerols(from Borrelia burgdorferi), lypophosphoglycan (from Leishmaniadonovani), endogenous or exogenous β-glucosylceramide, andphosphatidylinositol tetramannoside (PIM4) (from Mycobacterium leprae).For additional lipids and/or glycolipids useful as a CD1d-restrictedantigens, see V. Cerundolo et al., “Harnessing invariant NKT cells invaccination strategies.” Nature Rev Immun, 9:28-38 (2009).

“Tolerogenic immune response” means any immune response that can lead toimmune suppression or tolerization, such as against a CD1d-restrictedantigen or an APC presentable antigen (i.e., antigen presented byprofessional or non-professional antigen-presenting cells) or a cell,tissue, organ, etc. that expresses such an antigen. Such immuneresponses include any reduction, delay or inhibition in an undesiredimmune response specific to the antigen or cell, tissue, organ, etc.that expresses such antigen. Such immune responses also include anystimulation, production, induction, promotion or recruitment in adesired immune response specific to the antigen or cell, tissue, organ,etc. that expresses such antigen. Tolerogenic immune responses,therefore, include the absence of or reduction in an undesired immuneresponse to an antigen that can be mediated by antigen reactive cells aswell as the presence or promotion of suppressive cells. Tolerogenicimmune responses as provided herein include immunological tolerance. To“generate a tolerogenic immune response” refers to the generation of anyof the foregoing immune responses specific to an antigen or cell,tissue, organ, etc. that expresses such antigen. The tolerogenic immuneresponse can be the result of MHC Class I-restricted presentation and/orMHC Class II-restricted presentation and/or B cell presentation and/orpresentation by CD1d, etc.

Tolerogenic immune responses include any response that leads to thestimulation, induction, production or recruitment of iNKT cells, such asthose that produce IL-4 and/or IL-10. Tolerogenic immune responses alsoinclude any response that inhibits the stimulation, induction,production or recruitment of iNKT cells that produce IFN-γ. Tolerogenicimmune responses also include any response that leads to thestimulation, induction, production or recruitment of CD4+ Treg cellscells (e.g., such as CD4+ CD25highFoxP3+ Treg cells) and/or CD8+ Tregcells, which may be downstream effects of the compositions providedherein (e.g., downstream effects as a result of regulatory cytokineproduction). In some embodiments, the tolerogenic immune response, isone that results in the conversion to a regulatory phenotypecharacterized by the production, induction, stimulation or recruitmentof regulatory cells or the production of regulatory cytokines.

CD4+ Treg cells can express the transcription factor FoxP3 and inhibitinflammatory responses and auto-immune inflammatory diseases (Humanregulatory T cells in autoimmune diseases. Cvetanovich G L, Hafler D A.Curr Opin Immunol. 2010 December; 22(6):753-60. Regulatory T cells andautoimmunity. Vila J, Isaacs J D, Anderson A E. Curr Opin Hematol. 2009July; 16(4):274-9). Such cells also suppress T-cell help to B-cells andinduce tolerance to both self and foreign antigens (Therapeuticapproaches to allergy and autoimmunity based on FoxP3+ regulatory T-cellactivation and expansion. Miyara M, Wing K, Sakaguchi S. J Allergy ClinImmunol. 2009 April; 123(4):749-55). Additionally, FoxP3 may be inducedin other immune cells such as CD8+ T cells, macrophages and iNKT cells.CD4+ Treg cells recognize antigen when presented by Class II proteins onAPCs. CD8+ Treg cells, which recognize antigen presented by Class Ibmolecule Qa-1, can also suppress T-cell help to B-cells and result inactivation of antigen-specific suppression inducing tolerance to bothself and foreign antigens. Disruption of the interaction of Qa-1 withCD8+ Treg cells has been shown to dysregulate immune responses andresults in the development of auto-antibody formation and an auto-immunelethal systemic-lupus-erythematosus (Kim et al., Nature. 2010 Sep. 16,467 (7313): 328-32). CD8+ Treg cells have also been shown to inhibitmodels of autoimmune inflammatory diseases including rheumatoidarthritis and colitis (CD4+ CD25+ regulatory T cells in autoimmunearthritis. Oh S, Rankin A L, Caton A J. Immunol. Rev. 2010 January;233(1):97-111. Regulatory T cells in inflammatory bowel disease. Boden EK, Snapper S B. Curr Opin Gastroenterol. 2008 November; 24(6):733-41).In some embodiments, the compositions provided can effectively result inboth types of responses (CD4+ Treg and CD8+ Treg).

Tolerogenic immune responses also include, but are not limited to, theinduction of regulatory cytokines, such as Treg cytokines; induction ofinhibitory cytokines; the inhibition of inflammatory cytokines (e.g.,IL-4, IL-1b, IL-5, TNF-α, IL-6, GM-CSF, IFN-γ, IL-2, IL-9, IL-12, IL-17,IL-18, IL-21, IL-22, IL-23, M-CSF, C reactive protein, acute phaseprotein, chemokines (e.g., MCP-1, RANTES, MIP-1α, MIP-1β, MIG, ITAC orIP-10), the production of anti-inflammatory cytokines (e.g., IL-4,IL-13, IL-10, etc.), chemokines (e.g., CCL-2, CXCL8), proteases (e.g.,MMP-3, MMP-9), leukotrienes (e.g., CysLT-1, CysLT-2), prostaglandins(e.g., PGE2) or histamines; the inhibition of polarization to a Th17,Th1 or Th2 immune response; the inhibition of effector cell-specificcytokines: Th17 (e.g., IL-17, IL-25), Th1 (IFN-γ), Th2 (e.g., IL-4,IL-13); the inhibition of Th1-, Th2- or Th17-specific transcriptionfactors; the inhibition of proliferation of effector T cells; theinduction of apoptosis of effector T cells; the induction of tolerogenicdendritic cell-specific genes; the induction of FoxP3 expression; theinhibition of IgE induction or IgE-mediated immune responses; theinhibition of antibody responses (e.g., antigen-specific antibodyproduction); the inhibition of T helper cell response; the production ofTGF-β and/or IL-10; the inhibition of effector function ofautoantibodies (e.g., inhibition in the depletion of cells, cell ortissue damage or complement activation); etc. In some embodiments, thetolerogenic immune response is the stimulation of iNKT cells. AssessingiNKT cell stimulation includes analyzing the phenotype, activation andcytokine production of iNKT cells.

Any of the foregoing may be measured in vivo in one or more animalmodels or may be measured in vitro. One of ordinary skill in the art isfamiliar with such in vivo or in vitro measurements. Undesired immuneresponses or tolerogenic immune responses can be monitored using, forexample, methods of assessing immune cell number and/or function,tetramer analysis, ELISPOT, flow cytometry-based analysis of cytokineexpression, cytokine secretion, cytokine expression profiling, geneexpression profiling, protein expression profiling, analysis of cellsurface markers, PCR-based detection of immune cell receptor gene usage(see T. Clay et al., “Assays for Monitoring Cellular Immune Response toActive Immunotherapy of Cancer” Clinical Cancer Research 7:1127-1135(2001)), etc. Undesired immune responses or tolerogenic immune responsesmay also be monitored using, for example, methods of assessing proteinlevels in plasma or serum, immune cell proliferation and/or functionalassays, etc. In some embodiments, tolerogenic immune responses can bemonitored by assessing the induction of FoxP3. In addition, specificmethods are described in more detail in the Examples.

Preferably, tolerogenic immune responses lead to the inhibition of thedevelopment, progression or pathology of the diseases, disorders orconditions described herein. Whether or not the inventive compositionscan lead to the inhibition of the development, progression or pathologyof the diseases, disorders or conditions described herein can bemeasured with animal models of such diseases, disorders or conditions.In some embodiments, the reduction of an undesired immune response orgeneration of a tolerogenic immune response may be assessed bydetermining clinical endpoints, clinical efficacy, clinical symptoms,disease biomarkers and/or clinical scores. Undesired immune responses ortolerogenic immune responses can also be assessed with diagnostic teststo assess the presence or absence of a disease, disorder or condition asprovided herein. Undesired immune responses can further be assessed bymethods of measuring proteins levels and/or function in a subject. Inembodiments, methods for monitoring or assessing undesired allergicresponses include assessing an allergic response in a subject by skinreactivity and/or allergen-specific antibody production.

In some embodiments, monitoring or assessing the generation of anundesired immune response or a tolerogenic immune response in a subjectcan be prior to the administration of a composition of syntheticnanocarriers provided herein and/or prior to administration of atransplantable graft or exposure to an allergen. In other embodiments,assessing the generation of an undesired immune response or tolerogenicimmune response can be after administration of a composition ofsynthetic nanocarriers provided herein and/or and after administrationof a transplantable graft or exposure to an allergen. In someembodiments, the assessment is done after administration of thecomposition of synthetic nanocarriers, but prior to administration of atransplantable graft or exposure to an allergen. In other embodiments,the assessment is done after administration of a transplantable graft orexposure to an allergen, but prior to administration of the composition.In still other embodiments, the assessment is performed prior to boththe administration of the synthetic nanocarriers and administration of atransplantable graft or exposure to an allergen, while in yet otherembodiments the assessment is performed after both the administration ofsynthetic nanocarriers and the administration of a transplantable graftor exposure to an allergen. In further embodiments, the assessment isperformed both prior to and after the administration of the syntheticnanocarriers and/or administration of a transplantable graft or exposureto an allergen. In still other embodiments, the assessment is performedmore than once on the subject to determine that a desirable immune stateis maintained in the subject, such as a subject that has or is at riskof having an inflammatory disease, an autoimmune disease, an allergy orgraft verus host disease. Other subjects include those that haveundergone or will undergo transplantation.

An antibody response can be assessed by determining one or more antibodytiters. “Antibody titer” means a measurable level of antibodyproduction. Methods for measuring antibody titers are known in the artand include Enzyme-linked Immunosorbent Assay (ELISA). In embodiments,the antibody response can be quantitated, for example, as the number ofantibodies, concentration of antibodies or titer. The values can beabsolute or they can be relative. Assays for quantifying an antibodyresponse include antibody capture assays, enzyme-linked immunosorbentassays (ELISAs), inhibition liquid phase absorption assays (ILPAAs),rocket immunoelectrophoresis (RIE) assays and line immunoelectrophoresis(LIE) assays. When an antibody response is compared to another antibodyresponse the same type of quantitative value (e.g., titer) and method ofmeasurement (e.g., ELISA) is preferably used to make the comparison.

An ELISA method for measuring an antibody titer, for example, a typicalsandwich ELISA, may consist of the following steps (i) preparing anELISA-plate coating material such that the antibody target of interestis coupled to a substrate polymer or other suitable material (ii)preparing the coating material in an aqueous solution (such as PBS) anddelivering the coating material solution to the wells of a multiwellplate for overnight deposition of the coating onto the multiwell plate(iii) thoroughly washing the multiwell plate with wash buffer (such as0.05% Tween-20 in PBS) to remove excess coating material (iv) blockingthe plate for nonspecific binding by applying a diluent solution (suchas 10% fetal bovine serum in PBS), (v) washing the blocking/diluentsolution from the plate with wash buffer (vi) diluting the serumsample(s) containing antibodies and appropriate standards (positivecontrols) with diluent as required to obtain a concentration thatsuitably saturates the ELISA response (vii) serially diluting the plasmasamples on the multiwell plate such to cover a range of concentrationssuitable for generating an ELISA response curve (viii) incubating theplate to provide for antibody-target binding (ix) washing the plate withwash buffer to remove antibodies not bound to antigen (x) adding anappropriate concentration of a secondary detection antibody in samediluent such as a biotin-coupled detection antibody capable of bindingthe primary antibody (xi) incubating the plate with the applieddetection antibody, followed by washing with wash buffer (xii) adding anenzyme such as streptavidin-HRP (horse radish peroxidase) that will bindto biotin found on biotinylated antibodies and incubating (xiii) washingthe multiwell plate (xiv) adding substrate(s) (such as TMB solution) tothe plate (xv) applying a stop solution (such as 2N sulfuric acid) whencolor development is complete (xvi) reading optical density of the platewells at a specific wavelength for the substrate (450 nm withsubtraction of readings at 570 nm) (xvi) applying a suitablemultiparameter curve fit to the data and defining half-maximal effectiveconcentration (EC50) as the concentration on the curve at which half themaximum OD value for the plate standards is achieved.

A “transplantable graft” refers to a biological material, such as cells,tissues and organs (in whole or in part) that can be administered to asubject. Transplantable grafts may be autografts, allografts, orxenografts of, for example, a biological material such as an organ,tissue, skin, bone, nerves, tendon, neurons, blood vessels, fat, cornea,pluripotent cells, differentiated cells (obtained or derived in vivo orin vitro), etc. In some embodiments, a transplantable graft is formed,for example, from cartilage, bone, extracellular matrix, or collagenmatrices. Transplantable grafts may also be single cells, suspensions ofcells and cells in tissues and organs that can be transplanted.Transplantable cells typically have a therapeutic function, for example,a function that is lacking or diminished in a recipient subject. Somenon-limiting examples of transplantable cells are βcells, hepatocytes,hematopoietic stem cells, neuronal stem cells, neurons, glial cells, ormyelinating cells. Transplantable cells can be cells that areunmodified, for example, cells obtained from a donor subject and usablein transplantation without any genetic or epigenetic modifications. Inother embodiments, transplantable cells can be modified cells, forexample, cells obtained from a subject having a genetic defect, in whichthe genetic defect has been corrected, or cells that are derived fromreprogrammed cells, for example, differentiated cells derived from cellsobtained from a subject.

“Transplantation” refers to the process of transferring (moving) atransplantable graft into a recipient subject (e.g., from a donorsubject, from an in vitro source (e.g., differentiated autologous orheterologous native or induced pluripotent cells)) and/or from onebodily location to another bodily location in the same subject.

“Undesired immune response” refers to any undesired immune response thatresults from exposure to an antigen, promotes or exacerbates a disease,disorder or condition provided herein (or a symptom thereof), or issymptomatic of a disease, disorder or condition provided herein. Suchimmune responses generally have a negative impact on a subject's healthor is symptomatic of a negative impact on a subject's health.

C. INVENTIVE COMPOSITIONS

Provided herein are tolerogenic synthetic nanocarrier compositionscomprising immunosuppressants and CD1d-restricted antigens and relatedmethods. Such compositions and methods are useful for reducing thegeneration of undesired immune responses and promoting the generation oftolerogenic immune responses by, for example, stimulating iNKT cells.The compositions provided may also include other antigens, such as APCpresentable antigens. The compositions provided may also includetransplantable grafts. The compositions may be administered to subjectsin which a tolerogenic immune response is desired. Such subjects includethose that have or are at risk of having an inflammatory disease, anautoimmune disease, sickle cell disease, fatty liver disease,spontaneous abortion, an allergy, organ or tissue rejection or graftversus host disease. Such subjects also include those that haveundergone or will undergo transplantation.

A wide variety of synthetic nanocarriers can be used according to theinvention. In some embodiments, synthetic nanocarriers are spheres orspheroids. In some embodiments, synthetic nanocarriers are flat orplate-shaped. In some embodiments, synthetic nanocarriers are cubes orcubic. In some embodiments, synthetic nanocarriers are ovals orellipses. In some embodiments, synthetic nanocarriers are cylinders,cones, or pyramids.

In some embodiments, it is desirable to use a population of syntheticnanocarriers that is relatively uniform in terms of size, shape, and/orcomposition so that each synthetic nanocarrier has similar properties.For example, at least 80%, at least 90%, or at least 95% of thesynthetic nanocarriers, based on the total number of syntheticnanocarriers, may have a minimum dimension or maximum dimension thatfalls within 5%, 10%, or 20% of the average diameter or averagedimension of the synthetic nanocarriers. In some embodiments, apopulation of synthetic nanocarriers may be heterogeneous with respectto size, shape, and/or composition.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g. a polymeric core) and the shell is a secondlayer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more lipids. In some embodiments, a synthetic nanocarrier maycomprise a liposome. In some embodiments, a synthetic nanocarrier maycomprise a lipid bilayer. In some embodiments, a synthetic nanocarriermay comprise a lipid monolayer. In some embodiments, a syntheticnanocarrier may comprise a micelle. In some embodiments, a syntheticnanocarrier may comprise a core comprising a polymeric matrix surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In someembodiments, a synthetic nanocarrier may comprise a non-polymeric core(e.g., metal particle, quantum dot, ceramic particle, bone particle,viral particle, proteins, nucleic acids, carbohydrates, etc.) surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In other embodiments, synthetic nanocarriers may comprise metalparticles, quantum dots, ceramic particles, etc. In some embodiments, anon-polymeric synthetic nanocarrier is an aggregate of non-polymericcomponents, such as an aggregate of metal atoms (e.g., gold atoms).

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, hydroxyethylstarch, carageenan, glycon, amylose,chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch,chitin, inulin, konjac, glucommannan, pustulan, heparin, hyaluronicacid, curdlan, and xanthan. In embodiments, the inventive syntheticnanocarriers do not comprise (or specifically exclude) carbohydrates,such as a polysaccharide. In certain embodiments, the carbohydrate maycomprise a carbohydrate derivative such as a sugar alcohol, includingbut not limited to mannitol, sorbitol, xylitol, erythritol, maltitol,and lactitol.

In some embodiments, synthetic nanocarriers can comprise one or morepolymers. In some embodiments, the synthetic nanocarriers comprise oneor more polymers that is a nonmethoxy-terminated, pluronic polymer. Insome embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or99% (weight/weight) of the polymers that make up the syntheticnanocarriers are non-methoxy-terminated, pluronic polymers. In someembodiments, all of the polymers that make up the synthetic nanocarriersare non-methoxy-terminated, pluronic polymers. In some embodiments, thesynthetic nanocarriers comprise one or more polymers that is anon-methoxy-terminated polymer. In some embodiments, at least 1%, 2%,3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of thepolymers that make up the synthetic nanocarriers arenonmethoxy-terminated polymers. In some embodiments, all of the polymersthat make up the synthetic nanocarriers are non-methoxy-terminatedpolymers. In some embodiments, the synthetic nanocarriers comprise oneor more polymers that do not comprise pluronic polymer. In someembodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%of the polymers that make up the synthetic nanocarriers do not comprisepluronic polymer. In some embodiments, all of the polymers that make upthe synthetic nanocarriers do not comprise pluronic polymer. In someembodiments, such a polymer can be surrounded by a coating layer (e.g.,liposome, lipid monolayer, micelle, etc.). In some embodiments, variouselements of the synthetic nanocarriers can be coupled with the polymer.

The immunosuppressants and/or antigens can be coupled to the syntheticnanocarriers by any of a number of methods. Generally, the coupling canbe a result of bonding between the immunosuppressants and/or antigensand the synthetic nanocarrier. This bonding can result in theimmunosuppressants and/or antigens being attached to the surface of thesynthetic nanocarrier and/or contained within (encapsulated) thesynthetic nanocarrier. In some embodiments, however, theimmunosuppressants and/or antigens are encapsulated by the syntheticnanocarrier as a result of the structure of the synthetic nanocarrierrather than bonding to the synthetic nanocarrier. In preferableembodiments, the synthetic nanocarrier comprises a polymer as providedherein, and the immunosuppressants and/or antigens are coupled to thepolymer.

When coupling occurs as a result of bonding between theimmunosuppressants and/or antigens and synthetic nanocarriers, thecoupling may occur via a coupling moiety. A coupling moiety can be anymoiety through which an immunosuppressant and/or antigen is bonded to asynthetic nanocarrier. Such moieties include covalent bonds, such as anamide bond or ester bond, as well as separate molecules that bond(covalently or non-covalently) the immunosuppressant and/or antigen tothe synthetic nanocarrier. Such molecules include linkers or polymers ora unit thereof. For example, the coupling moiety can comprise a chargedpolymer to which an immunosuppressant and/or antigen electrostaticallybinds. As another example, the coupling moiety can comprise a polymer orunit thereof to which it is covalently bonded.

In preferred embodiments, the synthetic nanocarriers comprise a polymeras provided herein. These synthetic nanocarriers can be completelypolymeric or they can be a mix of polymers and other materials.

In some embodiments, the polymers of a synthetic nanocarrier associateto form a polymeric matrix. In some of these embodiments, a component,such as an immunosuppressant or antigen, can be covalently associatedwith one or more polymers of the polymeric matrix. In some embodiments,covalent association is mediated by a linker. In some embodiments, acomponent can be noncovalently associated with one or more polymers of apolymeric matrix. For example, in some embodiments a component can beencapsulated within, surrounded by, and/or dispersed throughout apolymeric matrix. Alternatively or additionally, a component can beassociated with one or more polymers of a polymeric matrix byhydrophobic interactions, charge interactions, van der Waals forces,etc. A wide variety of polymers and methods for forming polymericmatrices therefrom are known conventionally.

Polymers may be natural or unnatural (synthetic) polymers. Polymers maybe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers may be random, block, or comprise a combinationof random and block sequences. Typically, polymers in accordance withthe present invention are organic polymers.

In some embodiments, the polymer comprises a polyester, polycarbonate,polyamide, or polyether, or unit thereof. In other embodiments, thepolymer comprises poly(ethylene glycol) (PEG), polypropylene glycol,poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid),or a polycaprolactone, or unit thereof. In some embodiments, it ispreferred that the polymer is biodegradable. Therefore, in theseembodiments, it is preferred that if the polymer comprises a polyether,such as poly(ethylene glycol) or polypropylene glycol or unit thereof,the polymer comprises a block-co-polymer of a polyether and abiodegradable polymer such that the polymer is biodegradable. In otherembodiments, the polymer does not solely comprise a polyether or unitthereof, such as poly(ethylene glycol) or polypropylene glycol or unitthereof.

Other examples of polymers suitable for use in the present inventioninclude, but are not limited to polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g.poly(β-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine,polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated (e.g. coupled) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments may be made using the general teachings of U.S. Pat. No.5,543,158 to Gref et al., or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc.Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines. In embodiments, the inventive synthetic nanocarriers maynot comprise (or may exclude) cationic polymers.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that inventive synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention.

Compositions according to the invention comprise synthetic nanocarriersin combination with pharmaceutically acceptable excipients, such aspreservatives, buffers, saline, or phosphate buffered saline. Thecompositions may be made using conventional pharmaceutical manufacturingand compounding techniques to arrive at useful dosage forms. In anembodiment, inventive synthetic nanocarriers are suspended in sterilesaline solution for injection together with a preservative.

In embodiments, when preparing synthetic nanocarriers as carriers,methods for coupling components to the synthetic nanocarriers may beuseful. If the component is a small molecule it may be of advantage toattach the component to a polymer prior to the assembly of the syntheticnanocarriers. In embodiments, it may also be an advantage to prepare thesynthetic nanocarriers with surface groups that are used to couple thecomponent to the synthetic nanocarrier through the use of these surfacegroups rather than attaching the component to a polymer and then usingthis polymer conjugate in the construction of synthetic nanocarriers.

In certain embodiments, the coupling can be a covalent linker. Inembodiments, peptides according to the invention can be covalentlycoupled to the external surface via a 1,2,3-triazole linker formed bythe 1,3-dipolar cycloaddition reaction of azido groups on the surface ofthe nanocarrier with antigen or immunosuppressant containing an alkynegroup or by the 1,3-dipolar cycloaddition reaction of alkynes on thesurface of the nanocarrier with components containing an azido group.Such cycloaddition reactions are preferably performed in the presence ofa Cu(I) catalyst along with a suitable Cu(I)-ligand and a reducing agentto reduce Cu(II) compound to catalytic active Cu(I) compound. ThisCu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be referredas the click reaction.

Additionally, the covalent coupling may comprise a covalent linker thatcomprises an amide linker, a disulfide linker, a thioether linker, ahydrazone linker, a hydrazide linker, an imine or oxime linker, an ureaor thiourea linker, an amidine linker, an amine linker, and asulfonamide linker.

An amide linker is formed via an amide bond between an amine on onecomponent with the carboxylic acid group of a second component such asthe nanocarrier. The amide bond in the linker can be made using any ofthe conventional amide bond forming reactions with suitably protectedamino acids or components and activated carboxylic acid suchN-hydroxysuccinimide-activated ester.

A disulfide linker is made via the formation of a disulfide (S—S) bondbetween two sulfur atoms of the form, for instance, of R1—S—S—R2. Adisulfide bond can be formed by thiol exchange of a component containingthiol/mercaptan group(—SH) with another activated thiol group on apolymer or nanocarrier or a nanocarrier containing thiol/mercaptangroups with a component containing activated thiol group.

A triazole linker, specifically a 1,2,3-triazole of the form

wherein R1 and R2 may be any chemical entities, is made by the1,3-dipolar cycloaddition reaction of an azide attached to a firstcomponent such as the nanocarrier with a terminal alkyne attached to asecond component such as the immunosuppressant or antigen. The1,3-dipolar cycloaddition reaction is performed with or without acatalyst, preferably with Cu(I)-catalyst, which links the two componentsthrough a 1,2,3-triazole function. This chemistry is described in detailby Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) andMeldal, et al, Chem. Rev., 2008, 108(8), 2952-3015 and is often referredto as a “click” reaction or CuAAC.

In embodiments, a polymer containing an azide or alkyne group, terminalto the polymer chain is prepared. This polymer is then used to prepare asynthetic nanocarrier in such a manner that a plurality of the alkyne orazide groups are positioned on the surface of that nanocarrier.Alternatively, the synthetic nanocarrier can be prepared by anotherroute, and subsequently functionalized with alkyne or azide groups. Thecomponent is prepared with the presence of either an alkyne (if thepolymer contains an azide) or an azide (if the polymer contains analkyne) group. The component is then allowed to react with thenanocarrier via the 1,3-dipolar cycloaddition reaction with or without acatalyst which covalently couples the component to the particle throughthe 1,4-disubstituted 1,2,3-triazole linker.

A thioether linker is made by the formation of a sulfur-carbon(thioether) bond in the form, for instance, of R1-S—R2. Thioether can bemade by either alkylation of a thiol/mercaptan (—SH) group on onecomponent such as the component with an alkylating group such as halideor epoxide on a second component such as the nanocarrier. Thioetherlinkers can also be formed by Michael addition of a thiol/mercaptangroup on one component to an electron-deficient alkene group on a secondcomponent such as a polymer containing a maleimide group or vinylsulfone group as the Michael acceptor. In another way, thioether linkerscan be prepared by the radical thiol-ene reaction of a thiol/mercaptangroup on one component with an alkene group on a second component suchas a polymer or nanocarrier.

A hydrazone linker is made by the reaction of a hydrazide group on onecomponent with an aldehyde/ketone group on the second component such asthe nanocarrier.

A hydrazide linker is formed by the reaction of a hydrazine group on onecomponent with a carboxylic acid group on the second component such asthe nanocarrier. Such reaction is generally performed using chemistrysimilar to the formation of amide bond where the carboxylic acid isactivated with an activating reagent.

An imine or oxime linker is formed by the reaction of an amine orN-alkoxyamine (or aminooxy) group on one component with an aldehyde orketone group on the second component such as the nanocarrier.

An urea or thiourea linker is prepared by the reaction of an amine groupon one component with an isocyanate or thioisocyanate group on thesecond component such as the nanocarrier.

An amidine linker is prepared by the reaction of an amine group on onecomponent with an imidoester group on the second component such as thenanocarrier.

An amine linker is made by the alkylation reaction of an amine group onone component with an alkylating group such as halide, epoxide, orsulfonate ester group on the second component such as the nanocarrier.Alternatively, an amine linker can also be made by reductive aminationof an amine group on one component with an aldehyde or ketone group onthe second component such as the nanocarrier with a suitable reducingreagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.

A sulfonamide linker is made by the reaction of an amine group on onecomponent with a sulfonyl halide (such as sulfonyl chloride) group onthe second component such as the nanocarrier.

A sulfone linker is made by Michael addition of a nucleophile to a vinylsulfone. Either the vinyl sulfone or the nucleophile may be on thesurface of the nanocarrier or attached to a component.

The component can also be conjugated to the nanocarrier via non-covalentconjugation methods. For example, a negative charged antigen orimmunosuppressant can be conjugated to a positive charged nanocarrierthrough electrostatic adsorption. A component containing a metal ligandcan also be conjugated to a nanocarrier containing a metal complex via ametal-ligand complex.

In embodiments, the component can be attached to a polymer, for examplepolylactic acid-block-polyethylene glycol, prior to the assembly of thesynthetic nanocarrier or the synthetic nanocarrier can be formed withreactive or activatible groups on its surface. In the latter case, thecomponent may be prepared with a group which is compatible with theattachment chemistry that is presented by the synthetic nanocarriers'surface. In other embodiments, a peptide component can be attached toVLPs or liposomes using a suitable linker. A linker is a compound orreagent that capable of coupling two molecules together. In anembodiment, the linker can be a homobifuntional or heterobifunctionalreagent as described in Hermanson 2008. For example, a VLP or liposomesynthetic nanocarrier containing a carboxylic group on the surface canbe treated with a homobifunctional linker, adipic dihydrazide (ADH), inthe presence of EDC to form the corresponding synthetic nanocarrier withthe ADH linker. The resulting ADH linked synthetic nanocarrier is thenconjugated with a peptide component containing an acid group via theother end of the ADH linker on NC to produce the corresponding VLP orliposome peptide conjugate.

For detailed descriptions of available conjugation methods, seeHermanson G T “Bioconjugate Techniques”, 2nd Edition Published byAcademic Press, Inc., 2008. In addition to covalent attachment thecomponent can be coupled by adsorption to a pre-formed syntheticnanocarrier or it can be coupled by encapsulation during the formationof the synthetic nanocarrier.

Any immunosuppressant as provided herein can be coupled to the syntheticnanocarrier. Immunosuppressants include, but are not limited to,statins; mTOR inhibitors, such as rapamycin or a rapamycin analog; TGF-βsignaling agents; TGF-β receptor agonists; histone deacetylase (HDAC)inhibitors; corticosteroids; inhibitors of mitochondrial function, suchas rotenone; P38 inhibitors; NF-κβ inhibitors; adenosine receptoragonists; prostaglandin E2 agonists; phosphodiesterase inhibitors, suchas phosphodiesterase 4 inhibitor; proteasome inhibitors; kinaseinhibitors; G-protein coupled receptor agonists; G-protein coupledreceptor antagonists; glucocorticoids; retinoids; cytokine inhibitors;cytokine receptor inhibitors; cytokine receptor activators; peroxisomeproliferator-activated receptor antagonists; peroxisomeproliferator-activated receptor agonists; histone deacetylaseinhibitors; calcineurin inhibitors; phosphatase inhibitors and oxidizedATPs. Immunosuppressants also include IDO, vitamin D3, cyclosporine A,aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine,6-mercaptopurine, aspirin, niflumic acid, estriol, tripolide,interleukins (e.g., IL-1, IL-10), cyclosporine A, siRNAs targetingcytokines or cytokine receptors and the like.

Examples of statins include atorvastatin (LIPITOR®, TORVAST®),cerivastatin, fluvastatin (LESCOL®, LESCOL® XL), lovastatin (MEVACOR®,ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®,PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin(CRESTOR®), and simvastatin (ZOCOR®, LIPEX®).

Examples of mTOR inhibitors include rapamycin and analogs thereof (e.g.,CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap),C16-(S)-butylsulfonamidorapamycin (C16-BSrap),C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry &Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235), chrysophanicacid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001),KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available fromSelleck, Houston, Tex., USA).

Examples of TGF-β signaling agents include TGF-β ligands (e.g., activinA, GDF1, GDF11, bone morphogenic proteins, nodal, TGF-βs) and theirreceptors (e.g., ACVR1B, ACVR1C, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B,TGFβRI, TGFβRII), R-SMADS/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4,SMADS, SMAD8), and ligand inhibitors (e.g, follistatin, noggin, chordin,DAN, lefty, LTBP1, THBS1, Decorin).

Examples of inhibitors of mitochondrial function include atractyloside(dipotassium salt), bongkrekic acid (triammonium salt), carbonyl cyanidem-chlorophenylhydrazone, carboxyatractyloside (e.g., from Atractylisgummifera), CGP-37157, (−)-Deguelin (e.g., from Mundulea sericea), F16,hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360,SFK1, and valinomycin (e.g., from Streptomyces fulvissimus)(EMD4Biosciences, USA).

Examples of P38 inhibitors include SB-203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole),SB-239063(trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl)imidazole),SB-220025(5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)),and ARRY-797.

Examples of NF (e.g., NK-κβ) inhibitors includeIFRD1,2-(1,8-naphthyridin-2-yl)-Phenol, 5-aminosalicylic acid, BAY11-7082, BAY 11-7085, CAPE (Caffeic Acid Phenethylester),diethylmaleate, IKK-2 Inhibitor IV, IMD 0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], NFκB Activation Inhibitor III, NF-κB ActivationInhibitor II, JSH-23, parthenolide, Phenylarsine Oxide (PAO), PPM-18,pyrrolidinedithiocarbamic acid ammonium salt, QNZ, RO 106-9920,rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamideJ, rocaglaol, (R)-MG-132, sodium salicylate, triptolide (PG490),wedelolactone.

Examples of adenosine receptor agonists include CGS-21680 and ATL-146e.

Examples of prostaglandin E2 agonists include E-Prostanoid 2 andE-Prostanoid 4.

Examples of phosphodiesterase inhibitors (non-selective and selectiveinhibitors) include caffeine, aminophylline, IBMX(3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline,theobromine, theophylline, methylated xanthines, vinpocetine, EHNA(erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone (PERFAN),milrinone, levosimendon, mesembrine, ibudilast, piclamilast, luteolin,drotaverine, roflumilast (DAXAS™, DALIRESP™), sildenafil (REVATION®,VIAGRA®), tadalafil (ADCIRCA®, CIALIS®), vardenafil (LEVITRA®, STAXYN®),udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolopyrimidin-7-1.

Examples of proteasome inhibitors include bortezomib, disulfuram,epigallocatechin-3-gallate, and salinosporamide A.

Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximab(ERBITUX®), imatinib (GLEEVEC®), trastuzumab (HERCEPTIN®), gefitinib(IRESSA®), ranibizumab (LUCENTIS®), pegaptanib, sorafenib, dasatinib,sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib,E7080, pazopanib, mubritinib.

Examples of glucocorticoids include hydrocortisone (cortisol), cortisoneacetate, prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate (DOCA), and aldosterone.

Examples of retinoids include retinol, retinal, tretinoin (retinoicacid, RETIN-A®), isotretinoin (ACCUTANE®, AMNESTEEM®, CLARAVIS®,SOTRET®), alitretinoin (PANRETIN®), etretinate (TEGISON) and itsmetabolite acitretin (SORIATANE®), tazarotene (TAZORAC®, AVAGE®,ZORAC®), bexarotene (TARGRETIN®), and adapalene (DIFFERIN®).

Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonist,IGFBP, TNF-BF, uromodulin, Alpha-2-Macroglobulin, Cyclosporin A,Pentamidine, and Pentoxifylline (PENTOPAK®, PENTOXIL®, TRENTAL®).

Examples of peroxisome proliferator-activated receptor antagonistsinclude GW9662, PPARγ antagonist II1, G335, T0070907 (EMD4Biosciences,USA).

Examples of peroxisome proliferator-activated receptor agonists includepioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY171883, PPARy activator, Fmoc-Leu, troglitazone, and WY-14643(EMD4Biosciences, USA).

Examples of histone deacetylase inhibitors include hydroxamic acids (orhydroxamates) such as trichostatin A, cyclic tetrapeptides (such astrapoxin B) and depsipeptides, benzamides, electrophilic ketones,aliphatic acid compounds such as phenylbutyrate and valproic acid,hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824,and panobinostat (LBH589), benzamides such as entinostat (MS-275),CI994, and mocetinostat (MGCD0103), nicotinamide, derivatives of NAD,dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.

Examples of calcineurin inhibitors include cyclosporine, pimecrolimus,voclosporin, and tacrolimus.

Examples of phosphatase inhibitors include BN82002 hydrochloride,CP-91149, calyculin A, cantharidic acid, cantharidin, cypermethrin,ethyl-3,4-dephostatin, fostriecin sodium salt, MAZ51,methyl-3,4-dephostatin, NSC 95397, norcantharidin, okadaic acid ammoniumsalt from prorocentrum concavum, okadaic acid, okadaic acid potassiumsalt, okadaic acid sodium salt, phenylarsine oxide, various phosphataseinhibitor cocktails, protein phosphatase 1C, protein phosphatase 2Ainhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2,sodium orthovanadate.

In some embodiments, antigens as described herein are coupled tosynthetic nanocarriers. In some embodiments, the antigens are coupled tothe same or different synthetic nanocarriers as to which theimmunosuppressants are coupled. In other embodiments, the antigens arenot coupled to any synthetic nanocarriers. Antigens include thosedescribed above including antigens associated with an inflammatorydisease, autoimmune disease, allergy, fatty liver disease, spontaneousabortion, organ or tissue rejection or graft versus host disease, atransplantable graft, etc. The full length antigens themselves can becoupled to the synthetic nanocarriers. Fragments or derivatives of anyof the foregoing that include epitopes can also be coupled to thesynthetic nanocarriers.

In some embodiments, a component, such as an antigen orimmunosuppressant, may be isolated. Isolated refers to the element beingseparated from its native environment and present in sufficientquantities to permit its identification or use. This means, for example,the element may be (i) selectively produced by expression cloning or(ii) purified as by chromatography or electrophoresis. Isolated elementsmay be, but need not be, substantially pure. Because an isolated elementmay be admixed with a pharmaceutically acceptable excipient in apharmaceutical preparation, the element may comprise only a smallpercentage by weight of the preparation. The element is nonethelessisolated in that it has been separated from the substances with which itmay be associated in living systems, i.e., isolated from other lipids orproteins. Any of the elements provided herein may be isolated. Any ofthe antigens provided herein can be included in the compositions inisolated form.

D. METHODS OF MAKING AND USING THE INVENTIVE COMPOSITIONS AND RELATEDMETHODS

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods as nanoprecipitation, flow focusing fluidic channels, spraydrying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755; U.S. Pat. Nos. 5,578,325 and6,007,845; P. Paolicelli et al., “Surface-modified PLGA-basedNanoparticles that can Efficiently Associate and Deliver Virus-likeParticles” Nanomedicine. 5(6):843-853 (2010)).

Various materials may be encapsulated into synthetic nanocarriers asdesirable using a variety of methods including but not limited to C.Astete et al., “Synthesis and characterization of PLGA nanoparticles” J.Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K.Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide)Nanoparticles: Preparation, Properties and Possible Applications in DrugDelivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymericnanoparticles” Nanomedicine 2:8-21 (2006); P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). Other methods suitable for encapsulating materials intosynthetic nanocarriers may be used, including without limitation methodsdisclosed in U.S. Pat. No. 6,632,671 to Unger Oct. 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe coupled to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve.

Elements (i.e., components) of the inventive synthetic nanocarriers(such as moieties of which an immunofeature surface is comprised,targeting moieties, polymeric matrices, antigens, immunosuppressants andthe like) may be coupled to the overall synthetic nanocarrier, e.g., byone or more covalent bonds, or may be coupled by means of one or morelinkers. Additional methods of functionalizing synthetic nanocarriersmay be adapted from Published US Patent Application 2006/0002852 toSaltzman et al., Published US Patent Application 2009/0028910 toDeSimone et al., or Published International Patent ApplicationWO/2008/127532 A1 to Murthy et al.

Alternatively or additionally, synthetic nanocarriers can be coupled tocomponents directly or indirectly via non-covalent interactions. Innon-covalent embodiments, the non-covalent coupling is mediated bynon-covalent interactions including but not limited to chargeinteractions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. Such couplingsmay be arranged to be on an external surface or an internal surface ofan inventive synthetic nanocarrier. In embodiments, encapsulation and/orabsorption is a form of coupling. In embodiments, the inventivesynthetic nanocarriers can be combined with an antigen by admixing inthe same vehicle or delivery vehicle system.

Populations of synthetic nanocarriers may be combined to formpharmaceutical dosage forms according to the present invention usingtraditional pharmaceutical mixing methods. These include liquid-liquidmixing in which two or more suspensions, each containing one or moresubsets of nanocarriers, are directly combined or are brought togethervia one or more vessels containing diluent. As synthetic nanocarriersmay also be produced or stored in a powder form, dry powder-powdermixing could be performed as could the re-suspension of two or morepowders in a common media. Depending on the properties of thenanocarriers and their interaction potentials, there may be advantagesconferred to one or another route of mixing.

Typical inventive compositions that comprise synthetic nanocarriers maycomprise inorganic or organic buffers (e.g., sodium or potassium saltsof phosphate, carbonate, acetate, or citrate) and pH adjustment agents(e.g., hydrochloric acid, sodium or potassium hydroxide, salts ofcitrate or acetate, amino acids and their salts) antioxidants (e.g.,ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20,polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxycholate),solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol,trehalose), osmotic adjustment agents (e.g., salts or sugars),antibacterial agents (e.g., benzoic acid, phenol, gentamicin),antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g.,thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers andviscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488,carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethyleneglycol, ethanol).

Compositions according to the invention comprise inventive syntheticnanocarriers in combination with pharmaceutically acceptable excipients.The compositions may be made using conventional pharmaceuticalmanufacturing and compounding techniques to arrive at useful dosageforms. Techniques suitable for use in practicing the present inventionmay be found in Handbook of Industrial Mixing: Science and Practice,Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta,2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of DosageForm Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill Livingstone.In an embodiment, inventive synthetic nanocarriers are suspended insterile saline solution for injection together with a preservative.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method may require attention to theproperties of the particular moieties being associated.

In some embodiments, inventive synthetic nanocarriers are manufacturedunder sterile conditions or are terminally sterilized. This can ensurethat resulting compositions are sterile and non-infectious, thusimproving safety when compared to non-sterile compositions. Thisprovides a valuable safety measure, especially when subjects receivingsynthetic nanocarriers have immune defects, are suffering frominfection, and/or are susceptible to infection. In some embodiments,inventive synthetic nanocarriers may be lyophilized and stored insuspension or as lyophilized powder depending on the formulationstrategy for extended periods without losing activity.

The compositions of the invention can be administered by a variety ofroutes, including but not limited to subcutaneous, intranasal, oral,intravenous, intraperitoneal, intramuscular, transmuco sal,transmucosal, sublingual, rectal, ophthalmic, pulmonary, intradermal,transdermal, transcutaneous or intradermal or by a combination of theseroutes. Routes of administration also include administration byinhalation or pulmonary aerosol. Techniques for preparing aerosoldelivery systems are well known to those of skill in the art (see, forexample, Sciarra and Cutie, “Aerosols,” in Remington's PharmaceuticalSciences, 18th edition, 1990, pp. 1694-1712; incorporated by reference).

The transplantable grafts provided as a cell-based therapy of theinvention may be administered by parenteral, intraarterial, intranasalor intravenous administration or by injection to lymph nodes or anteriorchamber of the eye or by local administration to an organ or tissue ofinterest. The administration may be by subcutaneous, intrathecal,intraventricular, intramuscular, intraperitoneal, intracoronary,intrapancreatic, intrahepatic or bronchial injection.

The compositions of the invention can be administered in effectiveamounts, such as the effective amounts described elsewhere herein. Dosesof dosage forms contain varying amounts of populations of syntheticnanocarriers and/or varying amounts of immunosuppressants and/orantigens, according to the invention. The amount of syntheticnanocarriers and/or immunosuppressants and/or antigens present in theinventive dosage forms can be varied according to the nature of theantigens, the therapeutic benefit to be accomplished, and other suchparameters. In embodiments, dose ranging studies can be conducted toestablish optimal therapeutic amount of the population of syntheticnanocarriers and the amount of immunosuppressants and/or antigens to bepresent in the dosage form. In embodiments, the synthetic nanocarriersand/or the immunosuppressants and/or antigens are present in the dosageform in an amount effective to generate a tolerogenic immune response tothe antigens upon administration to a subject. It may be possible todetermine amounts of the immunosuppressants and/or antigens effective togenerate a tolerogenic immune response using conventional dose rangingstudies and techniques in subjects. Inventive dosage forms may beadministered at a variety of frequencies. In a preferred embodiment, atleast one administration of the dosage form is sufficient to generate apharmacologically relevant response. In more preferred embodiment, atleast two administrations, at least three administrations, or at leastfour administrations, of the dosage form are utilized to ensure apharmacologically relevant response.

Prophylactic administration of the inventive compositions can beinitiated prior to the onset of disease, disorder or condition ortherapeutic administration can be initiated after a disorder, disorderor condition is established.

In some embodiments, administration of synthetic nanocarriers isundertaken e.g., prior to administration of a transplantable graft orexposure to an allergen. In exemplary embodiments, syntheticnanocarriers are administered at one or more times including, but notlimited to, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,1, or 0 days prior to administration of a transplantable graft orexposure to an allergen. In addition or alternatively, syntheticnanocarriers can be administered to a subject following administrationof a transplantable graft or exposure to an allergen. In exemplaryembodiments, synthetic nanocarriers are administered at one or moretimes including, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 20, 25, 30, etc. days following administration of atransplantable graft or exposure to an allergen.

In some embodiments, a maintenance dose (e.g., of a syntheticnanocarrier composition provided herein) is administered to a subjectafter an initial administration has resulted in a tolerogenic responsein the subject, for example to maintain the tolerogenic effect achievedafter the initial dose, to prevent an undesired immune reaction in thesubject, or to prevent the subject becoming a subject at risk ofexperiencing an undesired immune response or an undesired level of animmune response. In some embodiments, the maintenance dose is the samedose as the initial dose the subject received. In some embodiments, themaintenance dose is a lower dose than the initial dose. For example, insome embodiments, the maintenance dose is about ¾, about ⅔, about ½,about ⅓, about ¼, about ⅛, about 1/10, about 1/20, about 1/25, about1/50, about 1/100, about 1/1,000, about 1/10,000, about 1/100,000, orabout 1/1,000,000 (weight/weight) of the initial dose.

The compositions and methods described herein can be used to induce orenhance a tolerogenic immune response and/or to suppress, modulate,direct or redirect an undesired immune response for the purpose ofimmune suppression. The compositions and methods described herein can beused in the diagnosis, prophylaxis and/or treatment of diseases,disorders or conditions in which immune suppression (e.g., tolerogenicimmune response) would confer a treatment benefit. Such diseases,disorders or conditions include inflammatory diseases, autoimmunediseases, allergies, sickle cell disease, fatty liver disease,spontaneous abortion, organ or tissue rejection and graft versus hostdisease. The compositions and methods described herein can also be usedin subjects who have undergone or will undergo transplantation.

Autoimmune diseases include, but are not limited to, rheumatoidarthritis, multiple sclerosis, immune-mediated or Type I diabetesmellitus, inflammatory bowel disease (e.g., Crohn's disease orulcerative colitis), systemic lupus erythematosus, psoriasis,scleroderma, autoimmune thyroid disease, alopecia greata, Grave'sdisease, Guillain-Barré syndrome, celiac disease, Sjögren's syndrome,rheumatic fever, gastritis, autoimmune atrophic gastritis, autoimmunehepatitis, insulitis, oophoritis, orchitis, uveitis, phacogenic uveitis,myasthenia gravis, primary myxoedema, pernicious anemia, autoimmunehaemolytic anemia, Addison's disease, scleroderma, Goodpasture'ssyndrome, nephritis, for example, glomerulonephritis, psoriasis,pemphigus vulgaris, pemphigoid, sympathetic opthalmia, idiopathicthrombocylopenic purpura, idiopathic feucopenia, Wegener'sgranulomatosis and poly/dermatomyositis.

Some additional exemplary autoimmune diseases, associated autoantigens,and autoantibodies, which are contemplated for use in the invention, aredescribed in Table 1 below:

Autoantibody Type Autoantibody Autoantigen Autoimmune disease ordisorder Antinuclear Anti-SSA/Ro ribonucleoproteins Systemic lupuserythematosus, neonatal antibodies autoantibodies heart block, primarySjögren's syndrome Anti-La/SS-B ribonucleoproteins Primary Sjögren'ssyndrome autoantibodies Anti-centromere centromere CREST syndromeantibodies Anti-neuronal Ri[disambiguation Opsoclonus nuclear antibody-2needed] Anti-dsDNA double-stranded SLE DNA Anti-Jo1 histidine-tRNAInflammatory myopathy ligase Anti-Smith snRNP core proteins SLE Anti-Type I Systemic sclerosis (anti-Scl-70 antibodies) topoisomerasetopoisomerase antibodies Anti-histone histones SLE and Drug-inducedLE[2] antibodies Anti-p62 nucleoporin 62 Primary biliarycirrhosis[3][4][5] antibodies[3] Anti-sp100 Sp100 nuclear antibodies [4]antigen Anti-glycoprotein- nucleoporin 210 kDa 210 antibodies[5] Anti-Anti-tTG Coeliac disease transglutaminase Anti-eTG Dermatitisherpetiformis antibodies Anti-ganglioside ganglioside GQ1B Miller-FisherSyndrome antibodies ganglioside GD3 Acute motor axonal neuropathy (AMAN)ganglioside GM1 Multifocal motor neuropathy with conduction block (MMN)Anti-actin actin Coeliac disease anti-actin antibodies antibodiescorrelated with the level of intestinal damage [6][7] Liver kidneyAutoimmune hepatitis.[8] microsomal type 1 antibody Lupus anticoagulantAnti-thrombin thrombin Systemic lupus erythematosus antibodiesAnti-neutrophil phospholipid Antiphospholipid syndrome cytoplasmicc-ANCA proteins in Wegener's granulomatosis antibody neutrophilcytoplasm p-ANCA neutrophil Microscopic polyangiitis, Churg-Straussperinuclear syndrome, systemic vasculitides (non- specific) Rheumatoidfactor IgG Rheumatoid arthritis Anti-smooth muscle smooth muscle Chronicautoimmune hepatitis antibody Anti-mitochondrial mitochondria Primarybiliary cirrhosis[9] antibody Anti-SRP signal recognitionPolymyositis[10] particle exosome complex Scleromyositis nicotinicMyasthenia gravis acetylcholine receptor muscle-specific Myastheniagravis kinase (MUSK) Anti-VGCC voltage-gated Lambert-Eaton myasthenicsyndrome calcium channel (P/Q-type) thyroid peroxidase Hashimoto'sthyroiditis (microsomal) TSH receptor Graves' disease Hu Paraneoplasticcerebellar syndrome Yo (cerebellar Paraneoplastic cerebellar syndromePurkinje Cells) amphiphysin Stiff person syndrome, paraneoplasticcerebellar syndrome Anti-VGKC voltage-gated Limbic encephalitis, Isaac'sSyndrome potassium channel (autoimmune neuromyotonia) (VGKC) basalganglia Sydenham's chorea, paediatric autoimmune neuronsneuropsychiatric disease associated with Streptococcus (PANDAS)N-methyl-D- Encephalitis aspartate receptor (NMDA) glutamic acidDiabetes mellitus type 1, stiff person decarboxylase syndrome (GAD)aquaporin-4 Neuromyelitis optica (Devic's syndrome)

Inflammatory diseases include, but are not limited to, Alzheimer's,Ankylosing spondylitis, arthritis, asthma, atherosclerosis, Behcet'sdisease, chronic inflammatory demyelinating polyradiculoneuropathy,Crohn's disease, colitis, cystic fibrosis, dermatitis, diverticulitis,hepatitis, irritable bowel syndrome (IBS), lupus erythematous, musculardystrophy, nephritis, Parkinson's, shingles and ulcerative colitis.Inflammatory diseases also include, for example, cardiovascular disease,chronic obstructive pulmonary disease (COPD), bronchiectasis, chroniccholecystitis, tuberculosis, Hashimoto's thyroiditis, sepsis,sarcoidosis, silicosis and other pneumoconioses, and an implantedforeign body in a wound, but are not so limited. As used herein, theterm “sepsis” refers to a well-recognized clinical syndrome associatedwith a host's systemic inflammatory response to microbial invasion. Theterm “sepsis” as used herein refers to a condition that is typicallysignaled by fever or hypothermia, tachycardia, and tachypnea, and insevere instances can progress to hypotension, organ dysfunction, andeven death.

In some embodiments, the inflammatory disease is non-autoimmuneinflammatory bowel disease, post-surgical adhesions, coronary arterydisease, hepatic fibrosis, acute respiratory distress syndrome, acuteinflammatory pancreatitis, endoscopic retrogradecholangiopancreatography-induced pancreatitis, burns, atherogenesis ofcoronary, cerebral and peripheral arteries, appendicitis, cholecystitis,diverticulitis, visceral fibrotic disorders, wound healing, skinscarring disorders (keloids, hidradenitis suppurativa), granulomatousdisorders (sarcoidosis, primary biliary cirrhosis), asthma, pyodermagandrenosum, Sweet's syndrome, Behcet's disease, primary sclerosingcholangitis or an abscess. In some preferred embodiment the inflammatorydisease is inflammatory bowel disease (e.g., Crohn's disease orulcerative colitis).

In other embodiments, the inflammatory disease is an autoimmune disease.The autoimmune disease in some embodiments is rheumatoid arthritis,rheumatic fever, ulcerative colitis, Crohn's disease, autoimmuneinflammatory bowel disease, insulin-dependent diabetes mellitus,diabetes mellitus, juvenile diabetes, spontaneous autoimmune diabetes,gastritis, autoimmune atrophic gastritis, autoimmune hepatitis,thyroiditis, Hashimoto's thyroiditis, insulitis, oophoritis, orchitis,uveitis, phacogenic uveitis, multiple sclerosis, myasthenia gravis,primary myxoedema, thyrotoxicosis, pernicious anemia, autoimmunehaemolytic anemia, Addison's disease, Anklosing spondylitis,sarcoidosis, scleroderma, Goodpasture's syndrome, Guillain-Barresyndrome, Graves' disease, glomerulonephritis, psoriasis, pemphigusvulgaris, pemphigoid, excema, bulous pemiphigous, sympathetic opthalmia,idiopathic thrombocylopenic purpura, idiopathic feucopenia, Sjogren'ssyndrome, systemic sclerosis, Wegener's granulomatosis,poly/dermatomyositis, primary biliary cirrhosis, primary sclerosingcholangitis, lupus or systemic lupus erythematosus.

Graft versus host disease (GVHD) is a complication that can occur aftera pluripotent cell (e.g., stem cell) or bone marrow transplant in whichthe newly transplanted material results in an attack on the transplantrecipient's body. In some instances, GVHD takes place after a bloodtransfusion. Graft-versus-host-disease can be divided into acute andchronic forms. The acute or fulminant form of the disease (aGVHD) isnormally observed within the first 100 days post-transplant, and is amajor challenge to transplants owing to associated morbidity andmortality. The chronic form of graft-versus-host-disease (cGVHD)normally occurs after 100 days. The appearance of moderate to severecases of cGVHD adversely influences long-term survival.

EXAMPLES Example 1 Immune Response of Synthetic Nanocarriers withCoupled Rapamycin With and without Ovalbumin Peptide (323-339) Materials

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609).Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Catalogue # R1017). PLGA with a lactide:glycolideratio of 3:1 and an inherent viscosity of 0.75 dL/g was purchased fromSurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;Product Code 7525 DLG 7A). Polyvinyl alcohol (85-89% hydrolyzed) waspurchased from EMD Chemicals (Product Number 1.41350.1001).

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.

Solution 2: Rapamycin @ 50 mg/mL in methylene chloride. The solution wasprepared by dissolving rapamycin in pure methylene chloride.

Solution 3: PLGA @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLGA in pure methylene chloride.

Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

Method for Preparing Synthetic Nanocarrier Containing Rapamycin andOvalbumin (323-339)

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.2 mL), and solution 3(1.0 mL) in a small pressure tube and sonicating at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) was then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for the syntheticnanocarriers to form. A portion of the synthetic nanocarriers werewashed by transferring the synthetic nanocarrier suspension to acentrifuge tube and centrifuging at 21,000×g and 4° C. for one hour,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated, and the pellet wasre-suspended in phosphate buffered saline for a final syntheticnanocarrier dispersion of about 10 mg/mL.

The amounts of peptide and rapamycin in the synthetic nanocarriers weredetermined by HPLC analysis. The total dry-synthetic nanocarrier massper mL of suspension was determined by a gravimetric method.

Method for Synthetic Nanocarrier Containing Rapamycin

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining 0.13 M hydrochloric acid solution (0.2 mL), solution 2 (0.2mL), and solution 3 (1.0 mL) in a small pressure tube and sonicating at50% amplitude for 40 seconds using a Branson Digital Sonifier 250. Asecondary emulsion (W1/O1/W2) was then prepared by combining solution 4(3.0 mL) with the primary W1/O1 emulsion, vortexing for 10 s, andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for the syntheticnanocarriers to form. A portion of the synthetic nanocarriers werewashed by transferring the synthetic nanocarrier suspension to acentrifuge tube and centrifuging at 21,000×g and 4° C. for one hour,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated, and the pellet wasre-suspended in phosphate buffered saline for a final syntheticnanocarrier dispersion of about 10 mg/mL.

The amount of rapamycin in the synthetic nanocarrier was determined byHPLC analysis. The total dry-synthetic nanocarrier mass per mL ofsuspension was determined by a gravimetric method.

Method for Measuring Rapamycin Load

Approximately 3 mg of synthetic nanocarriers were collected andcentrifuged to separate supernatant from synthetic nanocarrier pellet.Acetonitrile was added to the pellet, and the sample was sonicated andcentrifuged to remove any insoluble material. The supernatant and pelletwere injected on RP-HPLC and absorbance was read at 278 nm. The μg foundin the pellet were used to calculate % entrapped (load), μg insupernatant and pellet were used to calculate total μg recovered.

Method for Measuring Ovalbumin (323-339) Load

Approximately 3 mg of synthetic nanocarriers were collected andcentrifuged to separate supernatant from synthetic nanocarrier pellet.Trifluoroethanol was added to the pellet and the sample was sonicated todissolve the polymer, 0.2% trifluoroacetic acid was added and sample wassonicated and then centrifuged to remove any insoluble material. Thesupernatant and pellet were injected on RP-HPLC and absorbance was readat 215 nm. The μg found in the pellet were used to calculate % entrapped(load), μg in supernatant and pellet were used to calculate total μgrecovered.

Antigen-Specific Tolerogenic Dendritic Cells (tDC) Activity on Treg CellDevelopment

The assay included the use of OTII mice which have a transgenic T-cellreceptor specific for an immune-dominant ovalbumin (323-339). In orderto create antigen-specific tDCs, CD11c+ splenocytes were isolated, andthe ovalbumin (323-339) peptide added in vitro at 1 μg/ml or no antigen.Soluble or nanocarrier-encapsulated rapamycin was then added to the DCsfor 2 hours which were then washed extensively to remove free rapamycinfrom the culture. Purified responder CD4+ CD25- cells were isolated fromDT11 mice and added to tDC at a 10:1 T to DC ratio. The mixture of tDCand OTII T-cells were then cultured for 4-5 days, and the frequency ofTreg cells (CD4+ CD25highFoxP3+) were analyzed by flow cytometry asshown in FIG. 1. Regions were selected based on isotype controls.

Example 2 Mesoporous Silica Nanoparticles with Coupled Ibuprofen(Prophetic)

Mesoporous SiO2 nanoparticle cores are created through a sol-gelprocess. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g) is dissolvedin deionized water (500 mL), and then 2 M aqueous NaOH solution (3.5 mL)is added to the CTAB solution. The solution is stirred for 30 min, andthen Tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. Theresulting gel is stirred for 3 h at a temperature of 80° C. The whiteprecipitate which forms is captured by filtration, followed by washingwith deionized water and drying at room temperature. The remainingsurfactant is then extracted from the particles by suspension in anethanolic solution of HCl overnight. The particles are washed withethanol, centrifuged, and redispersed under ultrasonication. This washprocedure is repeated two additional times.

The SiO2 nanoparticles are then functionalized with amino groups using(3-aminopropyl)-triethoxysilane (APTMS). To do this, the particles aresuspended in ethanol (30 mL), and APTMS (50 μL) is added to thesuspension. The suspension is allowed to stand at room temperature for 2h and then is boiled for 4 h, keeping the volume constant byperiodically adding ethanol. Remaining reactants are removed by fivecycles of washing by centrifugation and redispersing in pure ethanol.

In a separate reaction, 1-4 nm diameter gold seeds are created. Allwater used in this reaction is first deionized and then distilled fromglass. Water (45.5 mL) is added to a 100 mL round-bottom flask. Whilestirring, 0.2 M aqueous NaOH (1.5 mL) is added, followed by a 1% aqueoussolution of tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL).Two minutes after the addition of THPC solution, a 10 mg/mL aqueoussolution of chloroauric acid (2 mL), which has been aged at least 15min, is added. The gold seeds are purified through dialysis againstwater.

To form the core-shell nanocarriers, the amino-functionalized SiO2nanoparticles formed above are first mixed with the gold seeds for 2 hat room temperature. The gold-decorated SiO2 particles are collectedthrough centrifugation and mixed with an aqueous solution of chloroauricacid and potassium bicarbonate to form the gold shell. The particles arethen washed by centrifugation and redispersed in water. Ibuprofen isloaded by suspending the particles in a solution of sodium ibuprofen (1mg/L) for 72 h. Free ibuprofen is then washed from the particles bycentrifugation and redispersing in water.

Example 3 Liposomes Containing Cyclosporine A (Prophetic)

The liposomes are formed using thin film hydration.1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol),cholesterol (32 μmol), and cyclosporin A (6.4 μmol) are dissolved inpure chloroform (3 mL). This lipid solution is added to a 50 mLround-bottom flask, and the solvent is evaporated on a rotary evaporatorat a temperature of 60° C. The flask is then flushed with nitrogen gasto remove remaining solvent. Phosphate buffered saline (2 mL) and fiveglass beads are added to the flask, and the lipid film is hydrated byshaking at 60° C. for 1 h to form a suspension. The suspension istransferred to a small pressure tube and sonicated at 60° C. for fourcycles of 30s pulses with a 30 s delay between each pulse. Thesuspension is then left undisturbed at room temperature for 2 h to allowfor complete hydration. The liposomes are washed by centrifugationfollowed by resuspension in fresh phosphate buffered saline.

Example 4 Polymeric Nanocarrier Containing Polymer-Rapamycin Conjugate(Prophetic)

Preparation of PLGA-rapamycin conjugate:

PLGA polymer with acid end group (7525 DLG1A, acid number 0.46 mmol/g,Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 eq) is dissolved in 30 mL ofdichloromethane (DCM). N,N-Dicyclohexylcarbodimide (1.2 eq, 2.8 mmol,0.57 g) is added followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and4-dimethylaminopyridine (DMAP) (2.0 eq, 4.6 mmol, 0.56 g). The mixtureis stirred at rt for 2 days. The mixture is then filtered to removeinsoluble dicyclohexylurea. The filtrate is concentrated to ca. 10 mL involume and added to 100 mL of isopropyl alcohol (IPA) to precipitate outthe PLGA-rapamycin conjugate. The IPA layer is removed and the polymeris then washed with 50 mL of IPA and 50 mL of methyl t-butyl ether(MTBE). The polymer is then dried under vacuum at 35 C for 2 days togive PLGA-rapamycin as a white solid (ca. 6.5 g).

Preparation of nanocarrier containing PLGA-rapamycin conjugate andovalbumin peptide (323-339):

Nanocarrier containing PLGA-rapamycin is prepared according to theprocedure described in Example 1 as follows:

Solutions for nanocarrier formation are prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution is prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.Solution 2: PLGA-rapamycin @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLGA-rapamycin in pure methylenechloride. Solution 3: PLA-PEG @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLA-PEG in pure methylene chloride.Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

A primary water-in-oil emulsion is prepared first. W1/O1 is prepared bycombining solution 1 (0.2 mL), solution 2 (0.75 mL), and solution 3(0.25 mL) in a small pressure tube and sonicating at 50% amplitude for40 seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) is then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250. TheW1/O1/W2 emulsion is added to a beaker containing 70 mM pH 8 phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers is washed by transferring thenanocarrier suspension to a centrifuge tube and centrifuging at 75,600×gand 4° C. for 35 min, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure is repeated,and the pellet is re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Example 5 Polymeric Nanocarrier Containing Polyamino Acid-LovastatinConjugate (Prophetic)

Preparation of poly-L-glutamic acid-lovastatin conjugate:Poly-L-glutamic acid sodium salt (5 g) (Alamanda Polymers, 30 kd MW, 200L-glutamic acid repeating unit) is dissolved in de-ionized water (30 mL)and the solution is cooled with ice water. Dilute HCl solution (1M) isadded with vigorous stirring while maintaining temperature below 10° C.until pH of the solution is about 2.5. The reaction mixture is warmed toroom temperature and stirred for 1 h. The suspension containinginsoluble poly-L-glutamic acid is centrifuged to remove the supernatant(aq. phase). The solid is then pellet washed with de-ionized water untilthe wash solution is at pH>3. The wet solid poly-L-glutamic acid is thenlyophilized to a constant weight as a white powder.

The poly-L-glutamic acid (2.0 g) is suspended in anhydrousN,N-diemthylformamide (DMF) (20 mL) with lovastatin (80 mg, 0.2 mmol)and DMAP (2 mmol, 0.24 g). The mixture is stirred at rt for 30 minutes.N,N-diisopropylcarbodiimide (2 mmol, 0.25 g) in 2 mL of DMF is addeddropwise. The reaction mixture is then stirred at rt overnight. Thereaction is cooled with icewater to <5 C and 10% NaCl solution is addedto slowly precipitate out the poly-L-glutamic acid-lovastatin conjugate.The precipitate is recovered by centrifuge. The wet solid is thenresuspended in water and the pH of the solution is adjusted to pH 7 with1M NaHCO3 solution. The resulting solution is stirred for 1 h at rt andfiltered through a 0.2 micron filter to remove impurities. The filtrateis cooled to <5° C. with ice water and HCl (1N) is added slowly withvigorous stirring until the pH of the solution to pH 3. After 30 min ofstirring, the precipitate solid is recovered by centrifuge. The solid isthe pellet washed twice with DI water and then lyophilized to givepoly-L-glutamic acid-lovastatin conjugate (ca. 1.5 g).

Preparation of nanocarrier containing poly-L-glutamic acid-lovastatinconjugate and ovalbumin peptide (323-339):

Nanocarrier containing poly-L-glutamic acid-lovastatin is preparedaccording to the procedure described in Example 1 as follows:

Solutions for nanocarrier formation are prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in 1×PBS buffer aqueoussolution. The solution is prepared by dissolving ovalbumin peptide in1×PBS buffer, pH 7.4 at room temperature. Solution 2: poly-L-glutamicacid-lovastatin @ 20 mg/mL in 1×PBS buffer. The solution is prepared bydissolving the polymer in 1×PBS buffer. Solution 3: PLGA @ 100 mg/mL inmethylene chloride. The solution was prepared by dissolving PLGA in puremethylene chloride. Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mMpH 7.4 phosphate buffer.

A primary water-in-oil emulsion is prepared first. W1/O1 is prepared bycombining solution 1 (0.2 mL), solution 2 (0.75 mL), and solution 3(0.25 mL) in a small pressure tube and sonicating at 50% amplitude for40 seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) is then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250. TheW1/O1/W2 emulsion is added to a beaker containing 70 mM pH 7.4 phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers is washed by transferring thenanocarrier suspension to a centrifuge tube and centrifuging at 75,600×gand 4° C. for 35 min, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure is repeated,and the pellet is re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Example 6 Preparation of Gold Nanocarriers (AuNCs) Containing Rapamycin(Prophetic)

Preparation of HS-PEG-rapamycin:

A solution of PEG acid disulfide (1.0 eq), rapamycin (2.0-2.5 eq), DCC(2.5 eq) and DMAP (3.0 eq) in dry DMF is stirred at rt overnight. Theinsoluble dicyclohexylurea is removed by filtration and the filtrate isadded to isopropyl alcohol (IPA) to precipitate out thePEG-disulfide-di-rapamycin ester and washed with IPA and dried. Thepolymer is then treated with tris(2-carboxyethyl)phosphine hydrochloridein DMF to reduce the PEG disulfide to thiol PEG rapamycin ester(HS-PEG-rapamycin). The resulting polymer is recovered by precipitationfrom IPA and dried as previously described and analyzed by H NMR andGPC.

Formation of Gold NCs (AuNCs):

An aq. solution of 500 mL of 1 mM HAuC14 is heated to reflux for 10 minwith vigorous stirring in a 1 L round-bottom flask equipped with acondenser. A solution of 50 mL of 40 mM of trisodium citrate is thenrapidly added to the stirring solution. The resulting deep wine redsolution is kept at reflux for 25-30 min and the heat is withdrawn andthe solution is cooled to room temperature. The solution is thenfiltered through a 0.8 μm membrane filter to give the AuNCs solution.The AuNCs are characterized using visible spectroscopy and transmissionelectron microscopy. The AuNCs are ca. 20 nm diameter capped by citratewith peak absorption at 520 nm.

AuNCs conjugate with HS-PEG-rapamycin:

A solution of 150 μl of HS-PEG-rapamycin (10 μM in 10 mM pH 9.0carbonate buffer) is added to 1 mL of 20 nm diameter citrate-capped goldnanocarriers (1.16 nM) to produce a molar ratio of thiol to gold of2500:1. The mixture is stirred at room temperature under argon for 1hour to allow complete exchange of thiol with citrate on the goldnanocarriers. The AuNCs with PEG-rapamycin on the surface is thenpurified by centrifuge at 12,000 g for 30 minutes. The supernatant isdecanted and the pellet containing AuNC—S-PEG-rapamycin is then pelletwashed with 1×PBS buffer. The purified Gold-PEG-rapamycin nanocarriersare then resuspend in suitable buffer for further analysis andbioassays.

Example 7 Evalutaing Tolerogenic Immune Response to Antigen In vivo(Prophetic)

A composition of the invention is dissolved in phosphate-buffered saline(PBS) or in PBS plus 0.02-0.5% Tween 20 in the case of lipids, andinjected into female Lewis rats intramuscularly in 0.1-0.2 ml containing500 μg of the composition. A control group of rats receives 0.1-0.2 mlof PBS+Tween alone. Within 5 hours after the injection, spleen and liverare harvested from the rats and single cell suspensions obtained bymacerating tissues through a 40 μm nylon cell strainer. For the liver,gradient centrifugation steps are performed to enrich the lymphocytefraction. Samples are stained in PBS (1% FCS) with the appropriatedilution of relevant monoclonal antibodies. Propidum iodide stainingcells are excluded from analysis. Samples are acquired on an LSR2 flowcytometer (BD Biosciences, USA) and analyzed using FACS Diva software.The expression of markers CD4, CD25high and FoxP3 is analyzed on iNKTcells. Also cells can be permebealized and intracellular cytokinestaining can be performed. The presence of CD4+ CD25highFoxP3+ cellssuggests an induction of Treg cells. The production of anti-inflammatorycytokines can suggest tolerizing actions of iNKT cells. This experimentcan also be performed 3 days following the injection and expansion ofiNKT cells and cytokine production can be measured.

Example 8 Evaluating Tolerogenic Immune Response by Invariant NaturalKiller T-Cell Phenotypic Analysis (Prophetic)

A composition of the invention is injected subcutaneously into femaleLewis rats. A control group of rats receives 0.1-0.2 ml of PBS. Nine toten days after the injection, spleen and lymph nodes are harvested fromthe rats and single cell suspensions obtained by macerating tissuesthrough a 40 μm nylon cell strainer. Samples are stained in PBS (1% FCS)with the appropriate dilution of relevant monoclonal antibodies.Propidium iodide staining cells are excluded from analysis. Samples areacquired on an LSR2 flow cytometer (BD Biosciences, USA) and analyzedusing FACS Diva software. Invariant natural killer T cells (iNKTs) areidentified by analyzing the expression of CD3, TCR and NK1.1 markers(such as NK1.1 or DX5) and specific T-cell receptor combinations(invariant Valpha and Vbeta chains). Invariant NKT cells can also bedetected with CD1d tetramers loaded with α-galactosylceramide (see,e.g., D. Li et al., Generation and characterization of CD1d tetramerproduced by a lentiviral expression system. J Immunol Methods. 2008 Jan.31; 330(1-2):57-63).

Example 9 Evaluating Tolerogenic Immune Response by AssessingInterleukin-10 Production (Prophetic)

A composition of the invention is injected subcutaneously into femaleLewis rats. A control group of rats receives 0.1-0.2 ml of PBS. Nine toten days after the injection, spleen and lymph nodes are harvested fromthe rats and single cell suspensions obtained by macerating tissuesthrough a 40 μm nylon cell strainer. Samples are stained in PBS (1% FCS)with the appropriate dilution of relevant monoclonal antibodies forsurface antigens. To stain intracellularly for IL-10, cells are fixedand permeabilized and IL-10 antibody is added for 30 min. Cells arewashed and propidium iodide staining cells are excluded from analysis.Samples are acquired on an LSR2 flow cytometer (BD Biosciences, USA) andanalyzed using FACS Diva software. The IL-10 level or the number ofIL-10 producing cells is then compared to a reference level or areference number, for example, a level or a number obtained from the ratbefore the injection of the composition or a level or number observed orexpected in the control group of rats. An increase in the level of IL-10or in the number of IL-10 producing cells in the rats who received thecomposition of the invention suggests an induction of a tolerogenicresponse.

Example 10 Evaluating iNKT Cell Activation with Synthetic NanocarriersComprising Immunosuppressant Nanocarriers

α-Galactosyl Ceramide (KRN7000) was purchased from Avanti Polar Lipids,Inc. (700 Industrial Park Drive Alabaster, Ala. 35007-9105; Catalognumber 867000P). PLGA with a lactide:glycolide ratio of 1:1 and aninherent viscosity of 0.45 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211; ProductCode 5050 DLG 4.5A). Polyvinyl alcohol (85-89% hydrolyzed) was purchasedfrom EMD Chemicals (Product Number 1.41350.1001).

Method

Solutions were prepared as follows:

Solution 1: KRN7000 @ 2 mg/mL in dimethylsulfoxide (DMSO). The solutionwas prepared by dissolving the dry lipid in pure DMSO. Solution 2: PLGA@ 100 mg/mL in methylene chloride. The solution was prepared bydissolving the PLGA in pure methylene chloride. Solution 3: Polyvinylalcohol @ 50 mg/mL in 100 mM pH 8 phosphate buffer.

A water-in-oil emulsion, (W/O) was prepared by combining solution 1 (1mL), solution 2 (1 mL), solution 3 (3 mL) in a small pressure tube andsonicating at 30% amplitude for 60 seconds using a Branson DigitalSonifier model 250, with the pressure tube immersed in an ice waterbath. The W/O emulsion was then added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for thenanocarriers to form. A portion of the nanocarriers were washed bytransferring the nanocarrier suspension to a centrifuge tube andcentrifuging at 75,600×g at 4° C. for 45 min, removing the supernatant,and re-suspending the pellet in phosphate buffered saline. The washingprocedure was repeated, and the pellet was re-suspended in phosphatebuffered saline for a final nanocarrier dispersion of about 10 mg/mL.Nanocarrier size was determined by dynamic light scattering. The amountof KRN7000 in the nanocarrier is reported as the theoretical loadinggiven no loss on processing. The total dry-nanocarrier mass per mL ofsuspension (NP concentration), was determined by a gravimetric method.

KRN7000 Content Effective (Theoretical % NP concentration NanocarrierDiameter (nm) wt/wt) (mg/mL) 212 2.0 9.0

Immunization

Mice were designated to groups that received PBS alone, soluble aGC,NPaGC, sol aGC+solRAPA, sol aGC+NP RAPA, NPaGC+solRAPA or NPaGC+NP RAPA.Mice were injected at 9 am and at 9.30 AM received BrefeldinA i.v. toprevent release of intracellular cytokines produced. At 1 pm, mice weresacrificed and the liver was perfused with PBS and processed to obtain asingle cell suspension enriched for lymphocytes. Cells were stained withcell surface markers for iNKT cells (agc-loaded CD1d tetramers) and Tcells receptor (TCRb) and activation marker CD69. The cell suspensionwas then permeabilized and intracellular cytokine staining was performedfor Th1 cytokine IFN-γ, and Th2 cytokines IL-4 and the anti-inflammatorycytokine IL-10. Cells were acquired on a FacsCanto flow cytometer andanalyzed on by FlowJo.

Results

iNKT cells from mice that received aGC were activated as seen bycytokine production and upregulation of CD69 on their surface.Unexpectedly, it was found that iNKT cells were more strongly andrapidly activated when they received NP aGC compared to sol aGC, as seenby rapid downregulation of their TCR. When mice received RAPA or NP RAPAalone, there was downregulation of CD69, suggesting the iNKT cells wereless active. In addition, a Th2 cytokine skewing with NPaGC was usedcompared to sol aGC. iNKT cells produced significantly more IL-4 andIL-10 when activated with NP aGC compared to sol aGC. The results areshown in FIGS. 2-5.

1. A composition comprising: (i) a first population of syntheticnanocarriers coupled to immunosuppressants, and (ii) a second populationof synthetic nanocarriers coupled to CD1d-restricted antigens.
 2. Thecomposition of claim 1, wherein the first population and secondpopulation are the same population.
 3. The composition of claim 1,wherein the immunosuppressants comprise a statin, an mTOR inhibitor, aTGF-β signaling agent, a corticosteroid, an inhibitor of mitochondrialfunction, a P38 inhibitor, an NF-κβ inhibitor, an adenosine receptoragonist, a prostaglandin E2 agonist, a phosphodiesterasse 4 inhibitor,an HDAC inhibitor or a proteasome inhibitor.
 4. (canceled)
 5. Thecomposition of claim 1, wherein the CD1d-restricted antigens compriseglycolipids.
 6. The composition of claim 1, wherein the CD1d-restrictedantigens comprise α-galactosylceramide, β-glucosylceramide, α-linkedglycosphingolipid from Sphingomonas spp., galactosyl diaglycerol fromBorrelia burgdorferi, lypophosphoglycan from Leishmania orphosphatidylinositol tetramannoside from Mycobacterium leprae.
 7. Thecomposition of claim 1, wherein the composition is in an amounteffective to generate a tolerogenic immune response when administered toa subject. 8-9. (canceled)
 10. The composition of claim 1, wherein thecomposition further comprises APC presentable antigens. 11-13.(canceled)
 14. The composition of claim 10, wherein the APC presentableantigens are autoantigens, allergens, or are associated with aninflammatory disease, fatty liver disease, an autoimmune disease, sicklecell disease, spontaneous abortion organ or tissue rejection or graftversus host disease.
 15. The composition of claim 1, wherein the load ofthe immunosuppressants and/or CD1d-restricted antigens on average acrossthe first and/or second population of synthetic nanocarriers is between0.0001% and 50%.
 16. (canceled)
 17. The composition of claim 1, whereinthe synthetic nanocarriers of the first population and/or secondpopulation and/or third population comprise lipid nanoparticles,polymeric nanoparticles, metallic nanoparticles, surfactant-basedemulsions, dendrimers, buckyballs, nanowires, virus-like particles orpeptide or protein particles. 18-27. (canceled)
 28. The composition ofclaim 1, wherein the mean of a particle size distribution obtained usingdynamic light scattering of the synthetic nanocarriers of the firstand/or second and/or third population is a diameter greater than 100 nm.29-32. (canceled)
 33. The composition of claim 1, wherein the aspectratio of the synthetic nanocarriers of the first population and/orsecond population and/or third population is greater than 1:1, 1:1.2,1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.
 34. (canceled)
 35. A dosage formcomprising the composition of claim
 1. 36. A method comprisingadministering the composition of claim 1 to a subject.
 37. A methodcomprising: administering to a subject a composition comprising: (i) afirst population of synthetic nanocarriers coupled toimmunosuppressants, and (ii) a second population of syntheticnanocarriers coupled to CD1d-restricted antigens, wherein thecomposition is in an amount effective to generate a tolerogenic immuneresponse in the subject.
 38. A method comprising: generating atolerogenic immune response in a subject by administering a compositioncomprising: (i) a first population of synthetic nanocarriers coupled toimmunosuppressants, and (ii) a second population of syntheticnanocarriers coupled to CD1d-restricted antigens.
 39. A methodcomprising: administering a composition to a subject according to aprotocol that was previously shown to generate a tolerogenic immuneresponse in one or more test subjects; wherein the compositioncomprises: (i) a first population of synthetic nanocarriers coupled toimmunosuppressants, and (ii) a second population of syntheticnanocarriers coupled to CD1d-restricted antigens. 40-81. (canceled) 82.A method comprising: (i) producing a first population of syntheticnanocarriers coupled to immunosuppressants, and (ii) producing a secondpopulation of synthetic nanocarriers coupled to CD1d-restrictedantigens. 83-93. (canceled)
 94. A process for producing a composition ordosage form comprising the steps of: (i) coupling a first population ofsynthetic nanocarriers to immunosuppressants, and (ii) coupling a secondpopulation of synthetic nanocarriers to CD1d-restricted antigens. 95.(canceled)
 96. A composition or dosage form obtainable by the method ofclaim
 82. 97-100. (canceled)